Annealing control primer and its uses

ABSTRACT

The present invention relates to an annealing control primer for improving annealing specificity in nucleic acid amplification and its applications to all fields of nucleic acid amplification-involved technology. The present primer comprises (a) a 3′-end portion having a hybridizing nucleotide sequence substantially complementary to a site on a template nucleic acid to hybridize therewith; (b) a 5′-end portion having a pre-selected arbitrary nucleotide sequence; and (c) a regulator portion positioned between said 3′-end portion and said 5′-end portion comprising at least one universal base or non-discriminatory base analog, whereby said regulator portion is capable of regulating an annealing portion of said primer in association with annealing temperature.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to an annealing control primer andits applications. More particularly, the present invention relates to anannealing control primer for improving annealing specificity in nucleicacid amplification and its applications to all fields of nucleic acidamplification-involved technology.

[0003] 2. Description of the Related Art

[0004] Nucleic acid amplification is a pivotal process for a widevariety of methods in molecular biology, so that various amplificationmethods have been proposed. For example, Miller, H. I. et al. (WO89/06700) disclose a nucleic acid sequence amplification based on thehybridization of a promoter/primer sequence to a target single-strandedDNA (“ssDNA”) followed by transcription of many RNA copies of thesequence. Other known nucleic acid amplification procedures includetranscription-based amplification systems (Kwoh, D. et al., Proc. Natl.Acad. Sci. U.S.A., 86:1173(1989); and Gingeras T. R. et al., WO88/10315).

[0005] Schemes based on ligation of two or more oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, arealso known (Wu, D. Y. et al., Genomics 4:560 (1989)), which are called“Ligation Chain Reaction” (LCR).

[0006] Davey, C. et al. (European Pat. Appln. Publication No. 329,822)disclose a nucleic acid amplification process involving cyclicallysynthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-strandedDNA (dsDNA). The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from resultingDNA:RNA duplex by the action of ribonuclease H. The resultant ssDNA is asecond template for a second primer, which also includes the sequencesof an RNA polymerase promoter. This primer is then extended by DNApolymerase, resulting as a double-stranded DNA (“dsDNA”) molecule,having a sequence identical to that of the original RNA between theprimers and having additionally, at one end, a promoter sequence. Thispromoter sequence can be used by the appropriate RNA polymerase toproduce many RNA copies of the DNA. These copies can then re-enter thecycle leading to very rapid amplification.

[0007] The most predominant process for nucleic acid amplification knownas polymerase chain reaction (hereinafter referred to as “PCR”), isbased on repeated cycles of denaturation of double-stranded DNA,followed by oligonucleotide primer annealing to the DNA template, andprimer extension by a DNA polymerase (Mullis et al. U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159; Saiki et al. 1985). Theoligonucleotide primers used in PCR are designed to anneal to oppositestrands of the DNA, and are positioned so that the DNA polymerasecatalyzed extension product of one primer can serve as the templatestrand for the other primer. The PCR amplification process results inthe exponential increase of discrete DNA fragments whose length isdefined by the 5′ ends of the oligonucleotide primers.

[0008] The success in the nucleic acid amplifications, in particular PCRamplification, relies on the specificity with which a primer annealsonly to its target (and not non-target) sequences and therefore it isimportant to optimize this molecular interaction. Whether a primer cananneal only to its perfect complement or also to sequences that have oneor more mismatches, depends critically upon the annealing temperature.In general, the higher the annealing temperature, the more specificannealing of the primer to its perfect matched template and so thegreater the likelihood of only target sequence amplification can beaccomplished. The lower the temperature, the more mismatches betweentemplate and primer can be tolerated, leading to increased amplificationof non-target sequences. Adjusting the annealing temperature can alterthe specificity of pairing between template and primer. For examples, ifthere is no product, the temperature may be too high and can be reduced.If there are products in control where only one primer is present, thisindicates that the single primer is annealing to more than one region ofthe template. In this case, the annealing temperature should beincreased. Considering such effect of annealing temperature on primerannealing specificity, there remains a strong need for an annealingcontrol primer system which is capable of controlling primer annealingin accordance with annealing temperature to enhance primer annealingspecificity regardless of primer design.

[0009] In addition to annealing temperature, several “primer searchparameters” such as primer length, GC content and PCR product length(Dieffenbach et al., 1995) should be considered for primer annealingspecificity. If a primer, which satisfies all such parameters, wereemployed, primer annealing would be specified, resulting in thesignificant enhancement of primer annealing specificity during targetDNA amplification and the freedom from the problems such as backgroundsand non-specific products arising from primers used in the experiments.It is usual that well-designed primers can help avoid non-specificannealing and backgrounds as well as distinguish between cDNAs orgenomic templates in RNA-PCR.

[0010] Many approaches have been developed to improve primer annealingspecificity and therefore accomplish the amplification of the desiredproduct. Examples are touchdown PCR (Don et al., 1991), hot start PCR(D'Aquila et al., 1991), nested PCR (Mullis and Faloona, 1987) andbooster PCR (Ruano et al., 1989). Another alternative approaches havebeen also reported that various ‘enhancer’ compounds can improve thespecificity of PCR. The enhancer compounds include chemicals thatincrease the effective annealing temperature of the reaction, DNAbinding proteins and commercially available reagents. However, there isno ‘magic’ additive that will ensure the success in every PCR and it isvery tedious to test different additives under different conditions suchas annealing temperature. Although these approaches have contributed tothe improvement of primer annealing specificity in some cases, they havenot accessed fundamentally to a solution for the problems arising fromprimers used in the PCR amplification, such as non-specific products andhigh backgrounds.

[0011] In many cases, the primer sequence does not need to be a perfectcomplement to the template sequence. The region of the primer thatshould be perfectly matched to the template is the 3′-end because thisend is the region of the primer extended by the DNA polymerase and istherefore the most important for ensuring the specificity of annealingto the correct target sequence. The 5′-end of the primer is lessimportant in determining specificity of annealing to the target sequenceand can be modified to carry additional sequence such as restrictionsites and promoter sequences that are not complementary to the template(McPherson and Moller 2000). This notion is adapted to the design of theannealing control primers of this invention as described below.

[0012] PCR-based techniques have been widely used not only foramplification of a target DNA sequence but also for scientificapplications or methods in the fields of biological and medical researchsuch as Reverse transcriptase PCR (RT-PCR), Differential Display PCR(DD-PCR), Cloning of known or unknown genes by PCR, Rapid amplificationof EDNA ends (RACE) and PCR-based genomic analysis (McPherson andMoller, 2000). The followings are only representatives of PCRapplications.

[0013] Techniques designed to identify genes that are differentiallyregulated by cells under various physiological or experimentalconditions (for example, differentiation, carcinogenesis,pharmacological treatment) have become pivotal in modern biology. Onesuch method for screening differences in gene expression between variouscell types or between different stages of cell development with theavailability of PCR is known as Differential Display PCR (DD-PCR),described by Liang and Pardee in 1992. This method uses combinations of10-mer arbitrary primers with anchored cDNA primers and generatesfragments that originate mostly from the poly(A) tail and extend about50-600 nucleotide upstream. By combining 3′ anchored Oligo(dT) primersand short 5′ arbitrary primers, the subsets of the transcriptome areamplified, the resulting cDNA fragments are generally separated ondenaturing polyacrylamide gel and visualized autoradiographically.

[0014] Although this method is simple and rapid and only requires smallamounts of total RNA, there are a number of disadvantages in theconventional DD-PCR methods. The differential banding patterns are oftenonly poorly reproducible due to the use of short arbitrary primer sothat many laboratories have had difficulty in obtaining reproducibleresults with these methods. It has been shown that at least 40% of thedifferentially displayed bands are not reproducible between experimentseven in well-trained hands (Bauer el al., 1994). Furthermore, thepattern of differential expression often cannot be reproducible onNorthern blots and the percentage of these false positives can arise upto 90% (Sompayrac et al., 1995). As a modification used for analternative, the use of longer random primers of, e.g. 20 bases inlength does not satisfactorily solve the problem of reproducibility (Itoet al., 1994). There are another factors responsible for the relativelylow reproducibility of DD-PCR such as an insufficient amount of startingmaterial and very low concentration of dNTP (2-5 μM) employed to preparethe different banding patterns (Matz and Lukyanov, 1998). It is alsodifficult to detect rare transcripts with these methods (Matz andLukyanov, 1998). In addition, because the cDNA fragments obtained fromDD-PCR are short (typically 100-500 bp) and correspond to the 3′-end ofthe gene that represent mainly the 3′ untranslated region, they usuallydo not contain a large portion of the coding region. Therefore, thelabor-intensive full-length cDNA screening is needed unless significantsequence homology, information for gene classification and prediction offunction is obtained (Matz and Lukyanov, 1998).

[0015]Differential Display methods generally use radioactive detectiontechniques using denaturing polyacrylamide gels. The radioactivedetection of the reaction products restricts the use of this techniqueto laboratories with the appropriate equipment. Relatively long exposuretimes and problems with the isolation of interesting bands from thepolyacrylamide gels are additional drawbacks of Differential Displaytechnique. Although modified non-radioactive Differential Displaymethods have recently been described, which include silver staining(Gottschlich et al. 1997; Kociok et al., 1998), fluorescent-labeledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), the use of biotinylated primers (Korn et al.,1992; Tagle et al., 1993; Rosok et al., 1996) and ethidiumbromide-stained agarose gels (Rompf and Kahl, 1997; Jefferies et al.,1998; Gromova et al., 1999), these methods have met with only limitedsuccess. If the reaction products could be simply detected on ethidiumbromide-stained agarose gel and the results were reproducible andreliable, it would greatly ncrease the speed of DD-PCR analysis andavoid the use of radioactivity.

[0016] Another PCR-based approach called targeted differential displayuses an oligonucleotide primer that directs the amplification ofmultigene family members with conserved protein domains. Gene familiesare groups of genes which are often functionally characterized by aparticular type of function undertaken by the gene products in a celland which structurally have one or more conserved regions (domains) incommon. Examples of gene families include the MADS-box and the homeogenefamily as well as further transcription factor families. The cyclin,cytokine and globin gene families are examples of medical interest. TheProsite Database provides a list of proteins that have common domainsand sequence motifs. The oligonucleotide used in the PCR can either be aspecific primer that is used at a low annealing temperature or, as ismore often the case, a degenerate primer mixture for use at higherstringencies (Stone and Wharton, 1994). However, amplifications usingdegenerate primers can sometimes be problematic and may requireoptimization. It is important to keep the annealing temperature as highas possible to avoid extensive nonspecific amplification and a good ruleof thumb is to use 55° C. as a starting temperature. In general, it isdifficult to keep this rule because degenerate primers should bedesigned on the basis of amino acid sequences or conserved domainsequences as a precondition. In order to generate a satisfiedrelationship between degenerate primer and annealing temperature in thisapproach, it is required to use an annealing control primer which cantolerate the alternation of annealing temperature, particularly hightemperature such as 68° C. regardless of primer design.

[0017] Still another PCR-based technique is arbitrary primed PCR(AP-PCR) for RNA fingerprinting. One great strength of AP-PCR methods istheir simplicity (Welsh and McClelland, 1991; Williams et al., 1990).AP-PCR uses a single primer or a pair of primers, wherein the primersare 10-mers or 18-mers as longer primer. This method has previously beenused to provide DNA fingerprints of hybrid cell lines (Ledbetter et al.,1990) and particular genomic regions (Welsh and McClelland, 1990;Williams et al., 1990). It provides a very useful tool for genomeanalysis in bacteria, fungi and plant identification and populationstudies, where individual isolates can be compared rapidly. For example,they can be used as a tool to identify pathogens or the occurrence ofparticular strains or pathotypes. Commonly, AP-PCR uses a single primerto initiate DNA synthesis from regions of a template where the primermatches imperfectly. In order for this to work, the initial cycles haveto be performed at low stringency (37-50° C.), normally for the firstfive cycles, which allows primer annealing to imperfect sites throughoutthe genome. The stringency is then increased (55° C.) as for standardPCR amplification and the reaction is allowed for an additional 30-35cycles. AP-PCR is not recommended for use in such applications aspaternity testing where unequivocal results are demanded, becausenonparental products are occasionally produced. Although alternativeAP-PCR approaches including nested AP-PCR have been developed(McClelland et al., 1993; Ralph et al., 1993), the issue ofreproducibility is still of main concern. One concern is that thepatterns may vary from day to day or from lab to lab (see, e.g., Meunierand Grimont, 1993).

[0018] Still yet another PCR-based application is RACE (rapidamplification of cDNA end) technology. RACE is a procedure foramplification of cDNA regions corresponding to the 5′- or 3′-end of mRNA(Frohman et al., 1988) and it has been used to isolate rare transcriptssuccessfully. The gene-specific primer may be derived from sequence datafrom a partial cDNA, genomic exon or peptide. In 3′ RACE, the polyA tailof mRNA molecules is exploited as a priming site for PCR amplification.mRNAs are converted into cDNAs using reverse transcriptase and anOligo-dT primer as known in the art. The generated cDNAs can then bedirectly PCR amplified using a gene-specific primer and a primer thatanneals to the polyA region.

[0019] The same principle as 3′ RACE applies to 5′ RACE but there is nopolyA tail. Thus, 5′ RACE is made by tagging the 5′-end of a cDNA bymeans of different methods (Fromont-Racine et al., 1993; Schaefer, 1995;Franz et al., 1999). Most approaches for the 5′ RACE such ashomopolymeric tailing and ligation anchored tailing require a set ofenzymatic reactions after completion of first strand cDNA synthesis(Schaefer, 1995). Each enzymatic step has the potential to introducefailures and to destroy the integrity of the cDNA. Recently, analternative has been introduced, the so-called CapFinder approach(Chenchik et al., 1998; Chenchik et al. U.S. Pat. Nos. 5,962,271 and5,962,272). The technique relies on dual functions of the reversetranscriptases: one is the terminal transferase activity to addnon-templated nucleotides to the 3′-end of a cDNA and the other is thetemplate switching activity to switch a template to a second template.This property is utilized during the retroviral life cycle (Clark, 1988;Kulpa et al., 1997). Moloney murine leukemia virus (M-MLV) reversetranscriptase (RT) often adds three to four non-template-derivedcytosine residues to the 3′-end of newly synthesized cDNAs in thepresence of manganese or high magnesium (Schmidt and Mueller, 1999).This approach allows the amplification of full-length cDNAs because theM-MLV RT adds C residues preferentially to the cDNA if complete (capped)mRNA serves as template.

[0020] However, the CapFinder approach for 5′-RACE experiments could notbe free from background problems such as DNA smear arising from thecontamination of the CapFinder and Oligo-dT primers, which are used incDNA synthesis (Chenchik et al., 1998). Even residual amounts of theseprimers result in a high background because both ideally fit to allcDNAs present in the reaction mixture. In addition, 3′-RACE andfull-length cDNA amplification have the same background problems due tothe contamination of primers used for cDNA synthesis in which theygenerate non-specific products in PCR reaction (Chenchik et al., 1998).New approaches to overcome the problems above have been recentlyintroduced. One approach is step-out PCR to suppress unwanted PCRproducts (Matz et al., 1999) but it has been pointed out that thisapproach still remains a smear of DNA rather than a single DNA (Schrammet al., 2000). Another approach which is introduced more recently is touse solid-phase cDNA synthesis and procedures to remove all contaminantsused in cDNA synthesis (Schramm et al., 2000), but the major drawback ofthis technique is costly and time-consuming by requiring solid-phasecDNA synthesis and following procedures. Therefore, more effective,simple, rapid and inexpensive strategies are required to completelyeliminate problems arising from contamination of the primers such asOligo-dT or CapFinder primer used for cDNA synthesis.

[0021] In addition to RACE technologies, in current technologies forcDNA library construction, the 5′-ends of genes tend to beunder-represented in cDNA populations, especially where a poly(dT)primer is used during first cDNA strand synthesis and the startingmaterial is limited. Although a number of different approaches have beendeveloped to overcome this problem, most suffer from common limitationsproducing full-length cDNAs or 5′-enriched cDNAs with a number ofinherent problems. These approaches are complex or costly andtime-consuming by requiring multiple enzymatic steps and/or are notpronounced sensitive (Caminci et al., 1997; Suzuki et al., 1997; Guegleret al. U.S. Pat. Nos. 6,083,727 and 6,326,175; Hayashizaki. U.S. Pat.No. 6,143,528). Therefore, there is continued interest in thedevelopment of improved methods for generating full-length or5′-enriched cDNAs, particularly with the limited starting material.

[0022] Multiplex PCR is another variant of PCR in which more than onetarget sequence can be simultaneously amplified with more than one pairof primers in the same reaction. Since its first description in 1988(Chamberlain et al., 1988), this method has been successfully applied inmany areas of DNA testing, including analyses of gene deletion(Anonymous, 1992; Henegariu, et al., 1994), mutation and polymorphismanalysis (Shuber et al., 1993; Mutirangura et al., 1993), quantitativeanalysis (Zimmermann et al., 1996), and RNA detection (Zou et al.,1998). In the field of infection diseases, the technique has been shownto be a valuable method for identification of viruses, bacteria, fungi,and/or parasites.

[0023] However, the results obtained with multiplex PCR are frequentlycomplicated by the artifacts of the amplification procedure. Theseinclude “false-negative” results due to reaction failure and“false-positive” results such as the amplification of spurious products,which may be caused by annealing of the primers to sequences which arerelated to but distinct from the true recognition sequences. For use inmultiplex PCR, a primer should be designed so that its predictedhybridization kinetics are similar to those of the other primers used inthe sample multiplex reaction. While the annealing temperature andprimer concentrations may be calculated to some degree, the conditionsgenerally have to be empirically determined for each multiplex reaction.Since the possibility of non-specific priming increases with eachadditional primer pair, the conditions must be modified as necessary asindividual primer sets are added. Moreover, the artifacts that resultfrom competition for resources (e.g., depletion of primers) areaugmented in multiplex PCR, since the differences in the yields ofunequally amplified fragments are enhanced with each cycle. Thus, theoptimization of the reaction conditions for multiplex PCR can becomelabor-intensive and time-consuming. Since the different multiplex PCRsmay have unique reaction conditions, the development of new diagnostictests can become very costly.

[0024] Therefore, there is a need in the art for primers that allowmultiplex PCR reactions to be designed and carried out without elaborateoptimization steps, irrespective of the potentially divergent propertiesof the different primers used. Furthermore, there is a need in the artfor primers that allow multiplex PCR reactions that, under the samereaction conditions, simultaneously produce equivalent amounts of eachof many amplification products.

[0025] Single nucleotide polymorphisms (SNPs), the most common geneticvariations found in the human genome, are important markers foridentifying disease-associated loci and for pharmaco-genetic studies(Landegren et al., 1998; Roses, 2000). SNPs appear in the human genomewith an average of once every 1000 bp and totaling >3 million. A varietyof approaches have been used to detect SNPs. However, one of the keybottlenecks is the amplification of DNA. Most current assays include astep that produces many copies of a short segment of the sample DNAspanning each target SNP. This amplification is usually necessarybecause only small amounts of DNA can be harvested from typical clinicalsamples. Also, the amplification improves the signal-to-noise ratio ofthe assays, increasing the reliability of detection. Most genotypingtechniques accomplish this amplification using PCR. Most importantly,the specificity of PCR amplification is critical in the application ofPCR in the SNP genotyping. Therefore, it would be beneficial if themethods for improving PCR specificity are available and applied to thedevelopment of SNP genotyping assay. It would also be beneficial if suchmethods are capable of providing multiple analyses in a single assay(multiplex assays).

[0026] As described above, all these methods and techniques involvingnucleic acid amplification, in particular PCR amplification, could notbe completely free from the limitations and problems resulting from thenon-specificity of primers used in each method, such as false positives,poor reproducibility, high backgrounds and so on, although improvedapproaches to each method has been continuously introduced. Therefore,there remains a need of novel primer for improving annealing specificityand methods, which can give rise to true results.

[0027] Throughout this application, various patents and publications arereferenced and citations are provided in parentheses. The disclosure ofthese patents and publications in their entities are hereby incorporatedby references into this application in order to more fully describe thisinvention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

[0028] Endeavoring to resolve the problems of such conventional primerand various methods involving nucleic acid amplification, the presentinventor has developed a novel annealing control primer that can permitnucleic acid amplification with much higher specificity and itsunlimited applications in all fields of nucleic acid amplification-basedtechnology.

[0029] Accordingly, it is an object of this invention to provide anannealing control primer for improving annealing specificity in nucleicacid amplification.

[0030] It is another object of this invention to provide a method foramplifying a nucleic acid sequence from a DNA or a mixture of nucleicacids as template.

[0031] It is still another object of this invention to provide a methodfor selectively amplifying a target nucleic acid sequence from a DNA ora mixture of nucleic acids as template

[0032] It is further object of this invention to provide a method forselectively amplifying a target nucleic acid sequence from an mRNA.

[0033] It is still further object of this invention to provide a methodfor detecting DNA complementary to differentially expressed mRNA in twoor more nucleic acid samples.

[0034] It is another object of this invention to provide a method forrapidly amplifying a target cDNA fragment comprising a cDNA regioncorresponding to the 3′-end region of an mRNA.

[0035] It is still another object of this invention to provide a methodfor amplifying a target cDNA fragment comprising a cDNA regioncorresponding to the 5′-end region of an mRNA.

[0036] It is further object of this invention to provide a method foramplifying a population of full-length double-stranded cDNAscomplementary to mRNAs.

[0037] It is still further object of this invention to provide a methodfor amplifying 5′-enriched double-stranded cDNAs complementary to mRNAs.

[0038] It is another object of this invention to provide a method foramplifying more than one target nucleotide sequence simultaneously,

[0039] It is still another object of this invention to provide a methodfor producing a DNA fingerprint of gDNA. It is still another object ofthis invention to provide a method for producing a RNA fingerprint of anmRNA sample.

[0040] It is further object of this invention to provide a method foridentifying a conserved homology segment in a multigene family.

[0041] It is still further object of this invention to provide a methodfor identifying a nucleotide variation in a target nucleic acid.

[0042] It is another object of this invention to provide a method formutagenesis in a target nucleic acid.

[0043] It is still another object of this invention to provide a kitcomprising an annealing control primer.

[0044] It is further object of this invention to provide kits for avariety of methods involving nucleic acid amplification.

[0045] It is still further object of this invention to provide a use ofan annealing control primer for a process involving nucleic acidamplification.

[0046] Other objects and advantages of the present invention will becomeapparent from the detailed description to follow taken in conjugationwith the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047]FIGS. 1A and 1B show schematic representations for selectivelyamplifying a target nucleic acid of double-stranded DNA (1A) or mRNA(1B) using the ACP of the present invention.

[0048]FIGS. 2A and 2B show schematic representations for identifyingdifferentially expressed genes using the ACP of the present invention.

[0049]FIG. 3 shows a schematic representation for amplifying a targetcDNA fragment comprising 3′-end region corresponding to the 3′-end ofmRNA using the ACP of the present invention.

[0050]FIGS. 4A and 4B show schematic representations for amplifying atarget cDNA fragment comprising 5′-end region corresponding to the5′-end of mRNA using the ACP of the present invention. The Oligo dT (4A)or random primer (4B) is used as a first-strand cDNA synthesis primer.

[0051]FIG. 5 shows a schematic representation for amplifying full-lengthcDNA molecules complementary to the mRNA molecules using the ACP of thepresent invention.

[0052]FIG. 6 shows a schematic representation for amplifying 5′ enrichedcDNA molecules complementary to the mRNA molecules comprising the 5′-endinformation using the ACP of the present invention.

[0053]FIG. 7A shows a schematic representation for detecting singlenucleotide polymorphism (SNP) using the ACP of the present invention.

[0054]FIG. 7B shows another schematic representation for detectingsingle nucleotide polymorphism (SNP) using the ACP of the presentinvention.

[0055]FIG. 8 is an agarose gel photograph to show the effect of adeoxyinosine group positioned between the 3′- and 5′-end portions ofACP. The cDNA was amplified using total RNA isolated from conceptustissues at E4.5 (lanes 1 and 4), E11.5 (lanes 2 and 5), and E18.5 (lanes3 and 6), with a set of the dT₁₀-JYC2 (SEG ID NO. 29) and ACP10 (lanes1-3) (SEG ID NO. 13), and a set of the dT₁₀-ACP1 (SEG ID NO. 30) andACP10 (lanes 4-6), respectively.

[0056]FIG. 9 is an agarose gel photograph to show the effect ofdeoxyinosine residues positioned between the 3′- and 5′-end portions ofACP in association with the alteration of number of deoxyinosine duringPCR. The lanes 0, 2, 4, 6, and 8 represent the number of deoxyinosineresidues, respectively.

[0057]FIG. 10A is an agarose gel photograph to show the results of twostage PCR amplifications for Esx1 using a set of EsxN7 and EsxC6 primers(lane 1) and a set of EsxN7-ACP and EsxC6-ACP primers (lane 2).

[0058]FIG. 10B is an agarose gel photograph to show the results of twostage PCR amplifications for Esx1 using EsxN1 (lane 1), EsxC2 (lane 2),a set of EsxN1-ACP and EsxC2 (lane 3), and a set of EsxN1-ACP andEsxC2-ACP (lane 4).

[0059]FIG. 10C is an agarose gel photograph to show the results of twostage PCR amplifications for Esx1 using a set of EsxN3 and EsxC5 (lanesI and 2) and a set of EsxN3-ACP and EsxC5-ACP (lane 3).

[0060]FIG. 10D is an agrasoe gel photograph to show the results ofnon-stop two stage PCR amplifications for Esx1 using the primer EsxN1(lane 1), EsxC2 (lane 2), a pair of EsxN1 and EsxC2 (lane 3) and a pairof EsxN1-ACP and EsxC2-ACP (lane 4).

[0061]FIG. 11A is a photograph of agarose gels to show examples of theACP used for detecting differentially expressed mRNAs during embryonicdevelopment using different stages of mouse conceptus tissues. The cDNAswere amplified using total RNA isolated from conceptus tissues at E4.5(lane 1), E11.5 (lane 2), and E18.5 (lane 3), with a set of ACP3 (SEG IDNO. 3) and dT₁₀-ACP1. The bands indicated by arrows represent the cDNAfragments amplified from differentially expressed mRNAs. The numbers ofthe arrows indicate the cDNA fragments used as probes in the Northernblot analysis of FIG. 13.

[0062]FIG. 11B is a photograph of agarose gels to show examples of theACP used for detecting differentially expressed mRNAs during embryonicdevelopment using different stages of mouse conceptus tissues. The cDNAswere amplified using total RNA isolated from conceptus tissues at E4.5(lanes 1-2 and 7-8), E11.5 (lanes 3-4 and 9-10), and E18.5 (lanes 5-6and 11-12), with a set of ACP5 (SEG ID NO. 5) and dT₁₀-ACP1 (the lanes1-6), and a set of ACP8 (SEG ID NO. 8) and dT₁₀-ACP1 (lanes 7-12),respectively. The bands indicated by arrows represent the cDNA fragmentsamplified from differentially expressed mRNAs. The numbers of the arrowsindicate the cDNA fragments used as probes in the Northern blot analysisof FIG. 13.

[0063]FIG. 11C is an agarose gel photograph to show the amplified cDNAproducts obtained from different stages of mouse conceptus samples(E4.5: lanes 1 and 2; E11.5: lanes 3 and 4; E18.5: lanes 5 and 6) usinga set of ACP10 and dT₁₀-ACP primers.

[0064]FIG. 11D is an agarose gel photograph to show the amplified cDNAproducts obtained from different stages of mouse conceptus samples(E4.5: lanes 1 and 2; E11.5: lanes 3 and 4; E18.5: lanes 5 and 6) usinga set of ACP14 and T₁₀-ACP1 primers.

[0065]FIG. 12A is an agarose gel photograph to show the amplified cDNAproducts obtained from different stages of mouse conceptus samples(E4.5: lane 1; E11.5: lane 2; E18.5: lane 3) by one-stop two-stage PCRamplification using a set of ACP10 and JYC5-T₁₅-ACP primers.

[0066]FIG. 12B is an agarose gel photograph to show the amplified cDNAproducts obtained from different stages of mouse conceptus samples(E4.5: lane 1; El 1.5: lane 2; E18.5: lane 3) by non-stop two-stage PCRamplification using a set of ACP10 and JYC5-T₁₅-ACP primers.

[0067]FIG. 13 shows Northern blot analysis of six cDNA fragmentsamplified from differentially expressed mRNAs during embryonicdevelopment. The six ³²P-labeled fragments indicated by arrows in FIG.11 were used as probes for Northern blot analysis. The arrows 1, 2, 3,4, 5, and 6 are DEG1 (FIG. 13A), DEG3 (FIG. 13B), DEG2 (FIG. 13C), DEG8(FIG. 13D), DEG5 (FIG. 13E), and DEG7 (FIG. 13F), respectively, whereinthe results of the DEG sequence analysis are shown in Table 1. DEG2 (SEGID NO. 31) and DEG5 (SEG ID NO. 32) are turned out as novel genes (Table2). The control panels (the lower part of each panel) show each gelbefore blotting, stained with ethidium bromide and photographed under UVlight, demonstrating similar levels of 18S and 28S rRNA as a loadingcontrol.

[0068]FIG. 14 shows the expression patterns of a novel gene, DEG5, in afull stage of mouse conceptus. Northern blot analysis was performedusing the radio-labeled DEG5 cDNA fragment as a probe. Total RNA (20μg/lane) was prepared from mouse conceptuses at the gestation times asindicated. The control panel at the lower part shows a gel beforeblotting, stained with ethidium bromide and photographed under UV light,demonstrating similar levels of 18S and 28S rRNA as a loading control.

[0069]FIG. 15 is an agarose gel photograph to show the differencebetween the conventional 3′-RACE (lane 1) and the ACP-based 3 ′-RACE(lane 2) with regard to beta-actin 3 ′-RACE.

[0070]FIG. 16 is an agarose gel photograph to show the differencebetween CapFinder methods and ACP-based methods for mouse JunB (lanes 1and 2) and beta-actin 5′-RACE (lanes 3 and 4) using the conventionalprimer (lanes 1 and 3) and ACP (lanes 2 and 4), respectively

[0071]FIG. 17 is an agarose gel photograph to show the differencebetween CapFinder methods and ACP-based methods for mouse PLP-C alpha5′-RACE using the conventional primer (lane 1) and ACP (lanes 2, 3, and4), respectively.

[0072]FIG. 18 shows the results of virtual Northern analysis by theCapFinder methods or ACP-based methods for the amplification of mousefull-length GAPDH cDNA.

[0073]FIG. 19 shows agarose gel photographs to show the results ofgenomic fingerprintings of 7 mouse stains using two different sets ofarbitrary ACPs.

[0074]FIG. 20 shows agarose gel photographs to show the amplifiedproducts of multiplex PCR by the conventional methods (A) or ACP-basedmethods (B) for the amplification of three target nucleic acids.

[0075]FIG. 21 shows agarose gel photographs to show the amplifiedproducts of multiplex PCR by the conventional methods (A) or ACP-basedmethods (B and C) for the amplification of four target nucleic acids.The ACP-based multiplex was conducted by one-stop (B) or non-stop (C)two-stage PCR amplification.

[0076]FIG. 22 shows an agarose gel photograph to show the results ofallele-specific amplification for a SNP in exon 4 of the human TP53 geneusing ACP.

[0077]FIG. 23 shows six agarose gel photographs which show the resultsof allele-specific amplifications using ACPs for six additional SNPseach present in different gene such as Beta-2 adrenergic receptor(ADRB2) (A), Chemokine (c-c motif) receptor 5 (CCR5) (B), Interleukin 13receptor (C), Leukocyte adhesion molecule-1 (LAM-1) (D), Tachykininreceptor 3 (TACR3) (E), and Interleukin 1, beta (IL1B) (F).

DETAILED DESCRIPTION OF THIS INVETNION

[0078] The present invention is generally directed to (a) an annealingcontrol primer for the specificity of nucleic acid amplification and (b)its applications. The annealing control primer of this invention(hereinafter referred to as “ACP”) allows primer annealing to becontrolled in association with annealing temperature, such that thespecificity of nucleic acid amplification (in particular, PCR) can besignificantly improved. The principle of the ACP is based on thecomposition of an oligonucleotide primer having 3′- and 5′-ends distinctportions separated by at least one universal base or non-discriminatorybase. The present inventor has discovered that the universal base ornon-discriminatory base group positioned between the 3′- and 5′- endportions plays as a regulator in controlling primer annealing to atemplate nucleic acid in associated with annealing temperature duringnucleic acid amplification. The presence of universal base ornon-discriminatory base residue group positioned between the 3′- and 5′-end portions interrupts the annealing of the 5′-end portion as well aslimits primer annealing to the 3′-end portion at certain annealingtemperature, which results in dramatic improvement of annealingspecificity. A universal base group positioned between the 3′- and5′-end portions of ACP is designed to define each portion. For thesereasons, the ACP is fundamentally different from the conventionalprimers in terms of the function for improving primer annealingspecificity under a particular stringency conditions during nucleic acidamplification.

[0079] The ACP of this invention is significantly effective and widelyaccessible to nucleic acid amplification-based applications. Also,various problems related to primer annealing specificity in theconventional PCR techniques can be fundamentally solved by the ACP. Themain benefits to be obtained from the use of the ACP during nucleic acidamplification (particularly PCR) are as follows:

[0080] (a) since the presence of an universal base residue grouppositioned between the 3′- and 5′-end portions restricts primerannealing portion to the 3′-end portion under such conditions that the3′-end portion anneals to the template, the annealing sequence of aprimer can be precisely controlled, which make it possible to design aprimer with a desired number of annealing sequence. It is particularlyuseful when an annealing portion of a primer has to be limited (e.g.,single nucleotide polymorphism (SNP) genotyping, DNA microarraryscreening, and detection of differentially expressed genes);

[0081] (b) since the presence of an universal base residue grouppositioned between the 3′- and 5′-end portions interrupts the annealingof the 5′-end portion to the template under such conditions that the3′-end portion anneals to the template, eventually the 5′-end portionnot involved in the annealing provides the 3′-end portion with primerannealing specificity;

[0082] (c) the specificity of primer annealing is highly sensitiveenough to detect even a single-base mismatching. Thus, it isparticularly useful for the identification of a nucleotide variation ina target nucleic acid, including, for example, single nucleotidepolymorphisms and point mutations;

[0083] (d) ACP is capable of providing a primer with a high tolerance in“primer search parameters” for primer design such as primer length,annealing temperature, GC content, and PCR product length;

[0084] (e) ACP system provides two-stage PCR amplifications which allowthe products to be excluded from non-specific amplification;

[0085] (f) the efficiency of PCR amplification is increased, which makesit easier to detect rare mRNAs; and

[0086] (g) the reproducibility of PCR products is increased, which savesa great amount of time and cost.

[0087] Principle of ACP

[0088] In one aspect of this invention, there is provided an annealingcontrol primer for improving annealing specificity in nucleic acidamplification, which comprises: (a) a 3′-end portion having ahybridizing nucleotide sequence substantially complementary to a site ona template nucleic acid to hybridize therewith; (b) a 5′-end portionhaving a pre-selected arbitrary nucleotide sequence; and (c) a regulatorportion positioned between said 3′-end portion and said 5′-end portioncomprising at least one universal base or non-discriminatory baseanalog, whereby said regulator portion is capable of regulating anannealing portion of said primer in association with annealingtemperature.

[0089] The principle of ACP is based on the composition of anoligonucleotide primer having 3′- and 5′-end distinct portions separatedby a regulator portion comprising at least one universal base ornon-discriminatory base and the effect of the regulator portion on the3′- and 5′-end portions in the oligonucleotide primer. The presence ofthe regulator portion comprising at least one universal base ornon-discriminatory base between the 3′- and 5′-end portions of ACP actsas a main factor which is responsible for the improvement of primerannealing specificity.

[0090] The term “template” refers to nucleic acid. The term “nucleicacid” is a deoxyribonucleotide or ribonucleotide polymer in eithersingle or double-stranded form, including known analogs of naturalnucleotides unless otherwise indicated. Therefore, the ACP of thisinvention can be employed in nucleic acid amplification using single ordouble-stranded gDNA, cDNA or mRNA as template. The term “portion” usedherein in conjunction with the primer of this invention refers to anucleotide sequence separated by the regulator portion. The term “3′-endportion” or “5′-end portion” refers to a nucleotide sequence at the3′-end or 5′-end of the primer of this invention, respectively, which isseparated by the regulator portion.

[0091] The term “primer”0 as used herein refers to an oligonucleotide,whether occurring naturally or produced synthetically, which is capableof acting as a point of initiation of synthesis when placed underconditions in which synthesis of primer extension product which iscomplementary to a nucleic acid strand (template) is induced, i.e., inthe presence of nucleotides and an agent for polymerization such as DNApolymerase and at a suitable temperature and pH. The primer ispreferably single stranded for maximum efficiency in amplification.Preferably, the primer is an oligodeoxyribonucleotide. The primer ofthis invention can be comprised of naturally occurring dNMP (i.e., dAMP,dGM, dCMP and dTMP), modified nucleotide or non-natural nucleotide. Theprimer can also include ribonucleotides. The primer must be sufficientlylong to prime the synthesis of extension products in the presence of theagent for polymerization. The exact length of the primers will depend onmany factors, including temperature, application and source of primer.The term “annealing” or “priming” as used herein refers to theapposition of an oligodeoxynucleotide or nucleic acid to a templatenucleic acid, whereby said apposition enables the polymerase topolymerize nucleotides into a nucleic acid molecule which iscomplementary to the template nucleic acid or a portion thereof.

[0092] The 3′-end portion of ACP has a nucleotide sequence substantiallycomplementary to a site on a template nucleic acid molecule. The term“substantially complementary” in reference to primer is used herein tomean that the primer is sufficiently complementary to hybridizeselectively to a template nucleic acid sequence under the designatedannealing conditions, such that the annealed primer can be extended bypolymerase to form a complementary copy of the template. Therefore, thisterm has a different meaning from “perfectly complementary” or relatedterms thereof. It will be appreciated that the 3′-end portion of ACP canhave one or more mismatches to template to an extent that the ACP canserve as primer. Most preferably, the 3′-end portion of ACP has anucleotide sequence perfectly complementary to a site on a template,i.e., no mismatches.

[0093] The 3′-end portion of ACP may have a wide variety of nucleotidesequences depending on its applications as well as template sequence.For example, where the ACP is applied to the process involving reversetranscription such as differential display PCR, RACE, amplification offull-length cDNA, fingerprinting, identification of conserved homologysegment and the like, its 3′-end portion may have the nucleotidesequence which hybridizes to the polyadenosine (poly A) tail of an mRNA,preferably at least 8 deoxythymidine nucleotides, more preferably atleast 10 deoxythymidine nucleotides and the most preferably, at least 10contiguous deoxythymidine nucleotides. For the process involving reversetranscription as above, in one embodiment, the 3′-end portion of ACP hasat least 10 contiguous deoxythymidine nucleotides having 3′-V at its3′-end; in which V is one selected from the group consisting ofdeoxyadenosine, deoxycytidine and deoxyguanosine, in another embodiment,at least 10 contiguous deoxythymidine nucleotides having 3′-NV at its3′-end; in which V is one selected from the group consisting ofdeoxyadenosine, deoxycytidine and deoxyguanosine, and N is one selectedfrom the group consisting of deoxyadenosine, deoxythymidine,deoxycytidine and deoxyguanosine.

[0094] Furthermore, where the ACP is employed in amplification of atarget nucleic acid sequence, its 3′-end portion comprises a nucleotidesequence substantially complementary to a target sequence; indifferential display PCR, an arbitrary sequence substantiallycomplementary to a site in a cDNA from an mRNA; in RACE, a gene-specificsequence substantially complementary to a site in a cDNA from an mRNA;in amplification of 5′-enriched cDNAs, a random sequence of at least sixnucleotides substantially complementary to sites in mRNAs; inidentification of conserved homology segment, a nucleotide sequencesubstantially complementary to a consensus sequence found in a genefamily or degenerate sequence selected from a plurality of combinationsof nucleotides encoding a predetermined amino acid sequence; inidentification of a nucleotide variation (e.g., allelic site) in atarget nucleic acid, a nucleotide sequence comprising a nucleotidecomplementary to the corresponding nucleotide of a nucleotide variation;and in mutagenesis, a nucleotide sequence comprising at least onemismatch nucleotide to a target nucleic acid.

[0095] The term “arbitrary” nucleotide sequence is used herein to meanthe nucleotide sequence that is chosen without knowledge of the sequenceof the target nucleic acids to be amplified. The term arbitrary shouldnot to be confused with “random” in reference to primer which connotes aprimer composed of a random population of primers each of different andrandom sequence. The term “degenerate” sequence in conjunction with ACPfor identification of conserved homology segment refers to thenucleotide sequence that is deducted from amino acid sequence, so thatthe degenerate sequence can form a pool of the nucleotide sequences fromone amino acid sequence due to degeneracy of genetic codon.

[0096] According to a preferred embodiment of the ACP, the pre-selectedarbitrary nucleotide sequence of the 5′-end portion is substantially notcomplementary to any site on the template nucleic acid.

[0097] According to a preferred embodiment, the annealing control primerof this invention can be represented by a general formula (1) of5′-X_(p)-Y_(q)-Z_(r)-3′, wherein X_(p) represents the 5′-end portionhaving the pre-selected arbitrary nucleotide sequence substantially notcomplementary to any site on the template nucleic acid; Yq representsthe regulator portion comprising at least one universal base ornon-discriminatory base analog; Zr represents the 3′-end portion havinga nucleotide sequence substantially complementary to a site on thetemplate nucleic acid; wherein p, q and r represent the number ofnucleotides; and wherein X, Y and Z is deoxyribonucleotide orribonucleotide.

[0098] The regulator portion comprising at least one universal base ornon-discriminatory base analog is responsible for the main function ofACP in associated with alteration of annealing temperature duringnucleic acid amplification. The term “universal base ornon-discriminatory base analog” used herein refers to one capable offorming base pairs with each of the natural DNA/RNA bases with littlediscrimination between them.

[0099] It has been widely known that nucleotides at some ambiguouspositions of degenerate primers have been replaced by universal base ora non-discriminatory analogue such as deoxyinosine (Ohtsuka et al, 1985;Sakanari et al., 1989), 1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole(Nichols et al., 1994) and 5-nitroindole (Loakes and Brown, 1994) forsolving the design problems associated with the degenerate primersbecause such universal bases are capable of non-specifically basepairing with all four conventional bases. However, there has not beenany report that this universal base or a non-discriminatory analoguesuch as deoxyinosine, 1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrroleand 5-nitroindole is used to increase the specificity of primerannealing during PCR.

[0100] The presence of universal base such as deoxyinosine,1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole and 5-nitroindole in aprimer generates low annealing temperatures due to its weaker hydrogenbonding interactions in base pairing. As an extension of this theory,the present inventor has induced that the presence of the contiguousuniversal bases between the 3′-end and 5′-end of a primer could generatea region which has lower melting temperature, forms a boundary to eachof 3′-and 5′-end portions of the primer, and affect the annealing ofeach portion, respectively. This theory provides the basis of theannealing control primers of this invention.

[0101] In a preferred embodiment, the ACP contains at least 2 universalbase or non-discriminatory base analog residues between the 3′- and5′-end portion sequences, more preferably, at least 3 universal bases ornon-discriminatory base analogs. Advantageously, the universal baseresidues between the 3′- and 5′-end portion sequences can be up to 15residues in length. According to one embodiment, the ACP contains 2-15universal base or non-discriminatory base analog residues. Mostpreferably, the universal bases between the 3′- and 5′-end portionsequences are about 5 residues in length.

[0102] With reference to the optimum number of universal base, i.e., 5residues, the minimum number of universal base residues between the 3′-and 5′-end portions of ACP is preferred in order to interrupt theannealing of the 5′-end portion to the template during nucleic acidamplification at certain annealing temperature. It is very likely thatthe length of universal base in the sequence (8-10 bases) does not makea significant difference on its own function in ACP.

[0103] The use of universal base residues between the 3′- and 5′-endportion sequences is considered as a key feature in the presentinvention because it provides each portion (3′- and 5′-end) with adistinct annealing specificity in association with an annealingtemperature during nucleic acid amplification, e.g. PCR.

[0104] According to a preferred embodiment, the universal base ornon-discriminatory base analog in the regulator portion includesdeoxyinosine, inosine, 7-deaza-2′-deoxyinosine, 2-aza-2′-deoxyinosine,2′-OMe inosine, 2′-F inosine, deoxy 3-nitropyrrole, 3-nitropyrrole,2′-OMe 3-nitropyrrole, 2′-F 3-nitropyrrole,1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole, deoxy 5-nitroindole,5-nitroindole, 2′-OMe 5-nitroindole, 2′-F 5-nitroindole, deoxy4-nitrobenzimidazole, 4-nitrobenzimidazole, deoxy 4-aminobenzimidazole,4-aminobenzimidazole, deoxy nebularine, 2′-F nebularine, 2′-F4-nitrobenzimidazole, PNA-5-introindole, PNA-nebularine, PNA-inosine,PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole,morpholino-nebularine, morpholino-inosine,morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole,phosphoramidate-5-nitroindole, phosphoramidate-nebularine,phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole,phosphoramidate-3-nitropyrrole, 2′-0-methoxyethyl inosine,2′0-methoxyethyl nebularine, 2′-0-methoxyethyl 5-nitroindole,2′-0-methoxyethyl 4-nitrobenzimidazole, 2′-0-methoxyethyl 3-nitropyrroleand combinations thereof, but not limited to. More preferably, theuniversal base or non-discriminatory base analog is deoxyinosine,1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole or 5-nitroindole, mostpreferably, deoxyinosine.

[0105] The preferred length of an oligonucleotide primer, as usedherein, is determined from desired specificity of annealing and thenumber of oligonucleotides having the desired specificity that arerequired to hybridize to the template. For example, an oligonucleotideprimer of 20 nucleotides is more specific than an oligonucleotide primerof 10 nucleotides because the addition of each nucleotide to anoligonucleotide increases the annealing temperature of the primer to thetemplate.

[0106] The lengths of the 3′- and 5′-end portion sequences of the ACPmay vary and depend in part on the objective of each application usingACP. In a preferred embodiment, the 3′-end portion of ACP is at least 6nucleotides in length, which is considered a minimal requirement oflength for primer annealing. More preferably, the 3′-end portionsequence is from 10 to 25 nucleotides and can be up to 60 nucleotides inlength. In another embodiment, the 3′-end portion of ACP can includeribonucleotides as well as deoxyribonucleotides.

[0107] In another preferred embodiment, the 5′-end portion of ACPcontains at least 15 nucleotides in length, which is considered aminimal requirement of length for annealing under high stringentconditions. Preferably, the 5′-end portion sequence can be up to 60nucleotides in length. More preferably, the 5′-end portion sequence isfrom 6 to 50 nucleotides, most preferably, from 20 to 25 nucleotides inlength. The entire ACP is preferably from 35 to 50 nucleotides inlength, and can be up to 100 nucleotides in length.

[0108] The 5′-end portion of ACP has a pre-selected arbitrary nucleotidesequence substantially not complementary to any site on the templatenucleic acid and this nucleotide sequence can serves as a priming sitefor subsequent amplification. The term “pre-selected arbitrary”nucleotide sequence used herein refers as any defined or pre-selecteddeoxyribonucleotide, ribonucleotide, or mixed deoxyribonucleotidesequence which contains a particular sequence of natural or modifiednucleotides. In some embodiment, the pre-selected arbitrary nucleotidesequence of the 5′-end portion can be composed of a universal primersequence such as T3 promoter sequence, T7 promoter sequence, SP6promoter sequence, and M13 forward or reverse universal sequence. Usinga longer arbitrary sequence (about 25 to 60 bases) at the 5′-end portionof ACP reduces the efficiency of ACP, but shorter sequences (about 15 to17 bases) reduce the efficiency of annealing at high stringentconditions of ACP. It is also a key feature of the present invention touse a pre-selected arbitrary nucleotide sequence at the 5′-end portionof ACP as a priming site for subsequent amplification.

[0109] According to one embodiment of the present invention, somemodifications in the 5′-end portion of ACP can be made unless themodifications abolish the advantages of the ACP, i.e., improvement inannealing specificity. For example, the 5′-end portion can comprises asequence or sequences recognized by a restriction endonuclease(s), whichmakes it feasible to clone the amplified product into suitable vector.In addition, the 5′-end portion can comprises at least one nucleotidewith a label for detection or isolation of amplified product. Suitablelabels include, but not limited to, fluorophores, chromophores,chemiluminescers, magnetic particles, radioisotopes, mass labels,electron dense particles, enzymes, cofactors, substrates for enzymes andhaptens having specific binding partners, e.g., an antibody,streptavidin, biotin, digoxigenin and chelating group. The 5′-endportion also comprises bacteriophage RNA polymerase promoter region.

[0110] According to the preferred embodiment of this invention, the ACPis applied to PCR. More preferably, the PCR is performed under a firstand a second annealing temperature, i.e., under different stringentconditions. The first annealing temperature may be equal to or lowerthan the second annealing temperature and preferably, the secondannealing temperature is higher than the first annealing temperature. Inthe PCR process performed under two different annealing temperatures,i.e., two-stage PCR, the 3′-end of ACP is involved in annealing at thefirst annealing temperature and the 5′-end of ACP incorporated intoamplified product of first amplification stage serves as a priming siteat the second annealing temperature. In this case, the advantages of ACPwill be demonstrated in accordance with the following assumptions:

[0111] (1) since a regulator portion of ACP is composed of at least oneuniversal base or non-discriminatory analogue which has lower Tm thanother portion in ACP due to its weaker hydrogen bonding interactions inbase pairing, the regulator portion of ACP is not favorable in annealingto the template nucleic acid under the conditions that the 3′-endportion of ACP anneals to a site of the template at a first annealingtemperature. Consequently, the presence of a regulator portioncomprising at least one universal base or non-discriminatory analoguebetween the 3′- and 5′-end portions of ACP restricts primer annealingportion to the 3′-end portion at first annealing temperature;

[0112] (2) the 5′-end portion which is not involved in the annealingunder the first annealing temperature keeps bothering the annealing ofthe 3′-end portion to the template;

[0113] (3) thus, the strength in which the specific annealing of the3′-end portion sequence occurs is relatively stronger than the strengthin which non-specific annealing occurs, under the first annealingtemperature, which results in the improvement of primer annealingspecificity at the 3′-end portion;

[0114] (4) where the 5′-end portion comprises a pre-selected arbitrary,nucleotide sequence, the portion serves as a priming site at a secondannealing temperature, which is high stringency conditions and alsoshould be higher than the first annealing temperature, for subsequentamplification of reaction product generated from annealing and extensionof the 3′-end portion sequence; and

[0115] (5) consequently, only the reaction product generated fromannealing and extension of the 3′-end portion sequence can be amplifiedclose to the theoretical optimum of a two-fold increase of product foreach PCR cycle under the second annealing temperature.

[0116] Therefore, the 3′-end portion of ACP acts only as annealing siteto the template at the first annealing temperature and the 5′-endportion of ACP is used as a priming site at the second annealingtemperature for the subsequent amplification of the product generated bycontacting and extending the 3′-end portion of ACP to the template.

[0117] It may be appreciated that the ACP of the present invention isvery useful in a variety of primer-based nucleic acid amplificationmethods including the methods of Miller, H. I. (WO 89/06700) and Davey,C. et al. (EP 329,822), Ligase Chain Reaction (LCR, Wu, D. Y. et al.,Genomics 4:560 (1989)), Polymerease Ligase Chain Reaction (Barany, PCRMethods and Applic., 1:5-16(1991)), Gap-LCR (WO 90/01069), Repair ChainReaction (EP 439,182), 3SR (Kwoh et al., PNAS, USA, 86:1173(1989)) andNASBA (U.S. Pat. No. 5,130,238), but not limited to.

[0118] In another aspect of this invention, there is provided a kitcomprising the annealing control primer or the annealing control primerset according to the present invention. According to one embodiment ofthis invention, this kit further comprises a primer or a primer pairhaving a nucleotide sequence corresponding to the 5′-end portion of theACP; in case that the 5′-end portion comprises universal primersequence, it is more preferred that the kit comprises the universalprimers. The present kits may optionally include the reagents requiredfor performing PCR reactions such as buffers, DNA polymerase, DNApolymerase cofactors, and deoxyribonucleotide-5′-triphosphates.Optionally, the kits may also include various polynucleotide molecules,reverse transcriptase, various buffers and reagents, and antibodies thatinhibit DNA polymerase activity. The kits may also include reagentsnecessary for performing positive and negative control reactions.Optimal amounts of reagents to be used in a given reaction can bereadily determined by the skilled artisan having the benefit of thecurrent disclosure. The kits, typically, are adapted to contain inseparate packaging or compartments the constituents afore-described.

[0119] The ACP of the subject invention can be applied to a variety ofnucleic acid amplification-based technologies. Representative examplesto prove the effect of ACP are:

[0120] I. Application to amplifying a nucleic acid sequence;

[0121] II. Application to amplifying a target nucleic acid sequence;

[0122] III. Application to multiplex DNA amplification;

[0123] IV. Application to the identification of differentially expressedgenes;

[0124] V. Application to rapid amplification of cDNA ends (RACE);

[0125] VI. Application to amplifying full-length cDNA;

[0126] VII. Application to amplifying 5′-enriched cDNA;

[0127] VIII. Application to DNA or RNA fingerprinting;

[0128] IX. Application to the identification of conserved homologysegments in multigene families;

[0129] X. Application to identification of a nucleotide sequencevariation;

[0130] XI. Application to mutagenesis; and

[0131] XII. Other applications.

[0132] I. Application to Amplifying a (target) Nucleic Acid Sequence

[0133] In still another of this invention, there is provided a methodfor amplifying a nucleic acid sequence from a DNA or a mixture ofnucleic acids, comprising performing an amplification reaction usingprimers, characterized in that at least one primer is derived from anyone of ACP described above. Preferably, the primer according to thestructure of ACP is one having at its 3′end portion a hybridizingsequence substantially complementary to a region of the nucleic acidsequence to hybridize therewith.

[0134] In a specific embodiment of this method, there is provided amethod using two stage amplifications for amplifying a nucleic acidsequence from a DNA or a mixture of nucleic acids, which comprises:

[0135] (a) performing a first-stage amplification of the nucleic acidsequence at a first annealing temperature comprising at least two cyclesof primer annealing, primer extending and denaturing, using the primerpair of any one of the ACP described above each having at its 3′ endportion a hybridizing sequence substantially complementary to a regionof the nucleic acid sequence to hybridize therewith, under conditions inwhich each primer anneals to the region of the nucleic acid sequence,whereby the amplification product of the nucleic acid sequence isgenerated; and

[0136] (b) performing a second-stage amplification of the amplificationproduct generated from step (a) at a second annealing temperature, whichis high stringent conditions, comprising at least one cycle of primerannealing, primer extending and denaturing, using the same primers asused in step (a) or a primer pair each comprising a pre-selectedarbitrary nucleotide sequence corresponding to each 5′-end portion ofthe primers used in step (a), under conditions in which each primeranneals to the 3′- and 5′-ends of the amplification product,respectively, whereby the amplification product is re-amplified.

[0137] Where the method is applied to the amplification of a targetnucleic acid sequence, the primer pair used has at its 3′-end portion ahybridizing sequence substantially complementary to a region of thetarget nucleic acid sequence to hybridize therewith. Therefore, in afurther aspect of this invention, there is provided a method forselectively amplifying a target nucleic acid sequence from a DNA or amixture of nucleic acids, wherein the method comprises performing anamplification reaction using primers, characterized in that at least oneprimer is derived from the ACP described above. Preferably, the primeraccording to the structure of ACP is one having at its 3′end portion ahybridizing sequence substantially complementary to a region of thetarget nucleic acid sequence to hybridize therewith.

[0138] In a specific embodiment of this method, there is provided amethod using two stage amplifications for selectively amplifying atarget nucleic acid sequence from a DNA or a mixture of nucleic acids,which comprises:

[0139] (a) performing a first-stage amplification of the target nucleicacid sequence at a first annealing temperature comprising at least twocycles of primer annealing, primer extending and denaturing, using theprimer pair of any one of the ACP described above each having at its3′end portion a hybridizing sequence substantially complementary to aregion of the target nucleic acid sequence to hybridize therewith, underconditions in which each primer anneals to its target nucleotidesequence, whereby the amplification product of the target nucleotidesequence is generated; and

[0140] (b) performing a second-stage amplification of the amplificationproduct generated from step (a) at a second annealing temperature, whichis high stringent conditions, comprising at least one cycle of primerannealing, primer extending and denaturing, using the same primers asused in step (a) or a primer pair each comprising a pre-selectedarbitrary nucleotide sequence corresponding to each 5′-end portion ofthe primers used in step (a), under conditions in which each primeranneals to the 3′- and 5′-ends of the amplification product,respectively, whereby the amplification product is re-amplified.

[0141] Where the template for amplification is mRNA, the production ofcDNA is required prior to amplification. Therefore, in still furtheraspect of this invention, there is provided a method for selectivelyamplifying a target nucleic acid sequence from an mRNA, wherin themethod comprises reverse transcribing the mRNA and performing anamplification reaction using primers, characterized in that at least oneprimer is derived from the ACP described above. Preferably, the primeraccording to the structure of ACP is one having at its 3′end portion ahybridizing sequence substantially complementary to a region of thetarget nucleic acid sequence to hybridize therewith.

[0142] In a specific embodiment of this invention, there is provided amethod using two stage amplifications for selectively amplifying atarget nucleic acid sequence from an mRNA which comprises:

[0143] (a) contacting the mRNA with an oligonucleotide dT primer whichis hybridized to polyA tail of the mRNA under conditions sufficient fortemplate driven enzymatic deoxyribonucleic acid synthesis to occur;

[0144] (b) reverse transcribing the mRNA to which the oligonucloetide dTpirmer hybridizes to produce a first DNA strand that is complementary tothe mRNA to which the oligonucloetide dT pirmer hybridizes;

[0145] (c) performing a first-stage amplification of the target nucleicacid sequence from the first DNA strand obtained from step (b) at afirst annealing temperature comprising at least two cycles of primerannealing, primer extending and denaturing, using the primer pair of ACPdescribed above having at its 3′end portion a hybridizing sequencesubstantially complementary to a region of the target nucleic acidsequence to hybridize therewith, under conditions in which each primeranneals to its target nucleotide sequence, whereby the amplificationproduct of the target nucleotide sequence is generated; and

[0146] (d) performing a second-stage amplification of the amplificationproduct generated from step (c) at a second annealing temperature, whichis high stringent conditions, comprising at least one cycle of primerannealing, primer extending and denaturing, using the same primers asused in step (c) or a primer pair each comprising a pre-selectedarbitrary nucleotide sequence corresponding to each 5′-end portion ofthe primers used in step (c), under conditions in which each primeranneals to the 3′- and 5′-ends of the amplification product,respectively, whereby the amplification product is re-amplified.

[0147] Since the amplification methods of this invention employs the ACPof this invention, the common descriptions between them are omitted inorder to avoid the complexity of this specification leading to unduemultiplicity.

[0148] This application using ACP of the subject invention can providean improved method for selectively amplifying a target nucleic acidsequence from a nucleic acid or a mixture of nucleic acids (DNA or mRNA)by performing nucleic acid amplifications, preferably, PCR. Since theeffect of ACP provides the conventional primers with primer annealingspecificity regardless of “primer search parameters” for primer designsuch as primer length, annealing temperature, GC content and productlength, it is particularly recommended to use the ACP when theconventional primers used to amplify a target nucleic acid fragment aretoo sensitive to such parameters to generate specific nucleic acidamplification products.

[0149] A schematic representation for selectively amplifying a targetnucleic acid of double-stranded DNA using novel ACP system as describedabove is illustrated in FIG. 1A. FIG. 1B illustrates a schematicrepresentation for selectively amplifying a target nucleic acid of mRNAusing novel ACP system. Referring to FIGS. 1A and 1B, the presentmethods will be described in more detail.

[0150] The present methods for amplifying a nucleic acid sequence may becarried out in accordance with various primer-based nucleic acidamplifications known in the art. Preferably, the methods are carried outaccording to the two stage amplifications developed by the presentinventor, more preferably, the amplification is performed by polymerasechain reaction known in the art and most preferably, hot start PCRmethod.

[0151] The methods of the present invention, for amplifying a nucleicacid sequence can be used to amplify any desired nucleic acid molecule.Such molecules may be either DNA or RNA. The molecule may be in either adouble-stranded or single-stranded form, preferably, double-stranded.Where the nucleic acid as starting material is double-stranded, it ispreferred to render the two strands into a single-stranded, or partiallysingle-stranded, form. Methods known to separate strands includes, butnot limited to, heating, alkali, formamide, urea and glycoxal treatment,enzymatic methods (e.g., helicase action) and binding proteins. Forinstance, strand separation can be achieved by heating at temperatureranging from 80° C. to 105° C. General methods for accomplishing thistreatment are provided by Joseph Sambrook, et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.(200 1).

[0152] Where a mRNA is employed as starting material for amplification,a reverse transcription step is necessary prior to amplification,details of which are found in Joseph Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.(2001); and Noonan, K. F. et al., Nucleic Acids Res.16:10366 (1988)). For reverse transcription, an oligonucleotide dTprimer hybridizable to poly A tail of mRNA is used. The oligonucleotidedT primer is comprised of dTMPs, one or more of which may be replacedwith other dNMPs so long as the dT primer can serve as primer. Reversetranscription can be done with a reverse transcriptase that has RNase Hactivity. If one uses an enzyme having RNase H activity, it may bepossible to omit a separate RNase H digestion step, by carefullychoosing the reaction conditions.

[0153] The present methods do not require that the molecules to beamplified have any particular sequence or length. In particular, themolecules which may be amplified include any naturally occurringprocaryotic, eukaryotic (for example, protozoans and parasites, fungi,yeast, higher plants, lower and higher animals, including mammals andhumans) or viral (for example, Herpes viruses, HIV, influenza virus,Epstein-Barr virus, hepatitis virus, polio virus, etc.) or viroidnucleic acid. The nucleic acid molecule can also be any nucleic acidmolecule which has been or can be chemically synthesized. Thus, thenucleic acid sequence may or may not be found in nature. The ACP usedfor the present invention is hybridized or annealed to a region ontemplate so that double-stranded structure is formed. Conditions ofnucleic acid hybridization suitable for forming such double strandedstructures are described by Joseph Sambrook, et al., Molecular Cloning,A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.(2001) and Haymes, B. D., et al., Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).The sequence of the 3′-end portion of ACP needs not to exhibit precisecomplementarity, but need only to be substantially complementary insequence to be able to form a stable double-stranded structure. Thus,departures from complete complementarity are permissible, so long assuch departures are not sufficient to completely preclude hybridizationto form a double-stranded structure. Hybridization of ACP to a region ontemplate nucleic acid is a prerequisite for its template-dependentpolymerization with polymerases. Factors (see Joseph Sambrook, et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.(2001); and Haymes, B. D., et. al.,Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C.(1985)) which affect the base pairing of ACP to its complementarynucleic acids subsequently affect priming efficiency. The nucleotidecomposition of ACP can affect the temperature at which annealing isoptimal and therefore can affect its priming efficiency.

[0154] A variety of DNA polymerases can be used in the amplificationstep of the present methods, which includes “Klenow” fragment of E. coliDNA polymerase I, a thermostable DNA polymerase and bacteriophage T7 DNApolymerase. Preferably, the polymerase is a thermostable DNA polymerasesuch as may be obtained from a variety of bacterial species, includingThermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus fliformis,Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu).Many of these polymerases may be isolated from bacterium itself orobtained commercially. Polymerase to be used with the subject inventioncan also be obtained from cells which express high levels of the clonedgenes encoding the polymerase. When a polymerization reaction, is beingconducted, it is preferable to provide the components required for suchreaction in excess in the reaction vessel. Excess in reference tocomponents of the amplification reaction refers to an amount of eachcomponent such that the ability to achieve the desired amplification isnot substantially limited by the concentration of that component. It isdesirable to provide to the reaction mixture an amount of requiredcofactors such as Mg²⁺, and dATP, dCTP, dGTP and dTTP in sufficientquantity to support the degree of amplification desired.

[0155] All of the enzymes used in this amplification reaction may beactive under the same reaction conditions. Indeed, buffers exist inwhich all enzymes are near their optimal reaction conditions. Therefore,the amplification process of the present invention can be done in asingle reaction volume without any change of conditions such as additionof reactants.

[0156] It would be understood that the 5′-end portions of a set of ACPsused in the step of the first-stage amplification could compriseidentical or different sequences; if they are identical, one primercorresponding to the sequence of 5′-end portion will be used in the stepof the second-stage amplification, whereas if they are different, twoprimers each corresponding to the sequence of each 5′-end portion ofACPs will be used in the step of the second-stage amplification.

[0157] The present invention includes an alternative process forselectively amplifying a target nucleic acid fragment from a nucleicacid or a mixture using ACP, wherein a set of primers comprising an ACPand a conventional primer can be used in the first amplification step,instead of a set of ACP. The term “conventional primer” used hereinrefers to any primer having a structure different from ACP, especially,in terms of the presence of the regulator portion containing universalbase. In this case, the conventional primer is added only the firstamplification step with the ACP and only one pre-selected arbitraryprimer corresponding to the 5′-end portion sequence of the ACP is addedin the second amplification step. In preferred embodiment, thealternative process can be used when each 3′-portion of a pair of ACP tobe used in the first amplification step has different meltingtemperature (T_(m)). “T_(m)” refers to the temperature at which half theprimers are annealed to the target region.

[0158] Two amplification steps of the present methods (in case ofamplification from mRNA, including reverse transcritptation) areseparated only in time. The first-stage amplification should be followedby the second-stage amplification. It would be understood that thefirst-stage amplification reaction mixture could include the primerscorresponding to the 5′-end portion which will be used to anneal to thesequences of the 5′-end portions of the ACPs in the second-stageamplification, which means that the primers corresponding to the 5′-endportion can be added to the reaction mixture at the time of or after thefirst-stage amplification step.

[0159] As an alternative process, in the second-stage amplification stepthe complete sequences of the ACPs used in the first-stage amplificationstep, instead of the primers corresponding to the 5′-end portions of theACPs, can be used as primers at the high stringent conditions forre-amplifying the product generated from the first-stage amplificationstep, wherein the 3′- and 5′- ends of the product from the firstamplification step which is generated from annealing and extension ofthe 3′-end portion sequence of the set of ACP to the template nucleicacid at the low stringent conditions comprise the sequence orcomplementary sequence of ACP and also serve as perfect paring sites tothe set of ACP. In this view, this alternative process is preferredbecause this need not further add the primers corresponding to the5′-end portions of the ACPs to the reaction mixture at the time of orafter the first-stage amplification step. FIG. 1A also illustrates aschematic representation for selectively amplifying a target nucleicacid by the alternative process stated above.

[0160] Annealing or hybridization in the present methods is performedunder stringent conditions that allow for specific binding between anucleotide sequence and ACP. Such stringent conditions for annealingwill be sequence-dependent and varied depending on environmentalparameters. In the present methods, the second-stage amplification isgenerally performed under higher stringent conditions than thefirst-stage amplification.

[0161] In a preferred embodiment, the first annealing temperature rangesfrom about 30° C. to 68° C. for the first-stage amplification step, morepreferably, 40° C. to 65° C. It is preferred that the second annealingtemperature ranges from about 50° C. to 72° C. for the second-stageamplification. According to a more preferred embodiment, the firstannealing temperature is equal to or lower than the second annealingtemperature. The length or melting temperature (T_(m)) of the 3′-endportion sequence of ACP will determine the annealing temperature for thefirst-stage amplification. For example, in case that ACP comprises 10arbitrary nucleotides at the 3′-end portion, preferably, the annealingtemperature will be about between 45° C. and 55° C. for the first-stageamplification.

[0162] According to the present methods, the first-stage amplificationunder low stringent conditions is carried out for at least 2 cycles ofannealing, extending and denaturing to improve the specificity of primerannealing during the first-stage amplification, and through thesubsequent cycles, the second-stage amplification is processed moreeffectively under high stringent conditions. The first-stageamplification can be carried out up to 30 cycles. In a preferredembodiment, the first-stage amplification is carried out for 2 cycles.In another embodiment, the second-stage amplification under highstringent conditions is carried out for at least one cycle (preferably,at least 5 cycles) and up to 45 cycles to amplify the first-stageproduct. In a more preferred embodiment, the second-stage amplificationis carried out for 25-35 cycles. High and low stringent conditions maybe readily determined from the standard known in the art. “Cycle” refersto the process which results in the production of a copy of targetnucleic acid. A cycle includes a denaturing step, an annealing step, andan extending step.

[0163] In the most preferable embodiment, the amplification is performedin accordance with PCR which is disclosed in U.S. Pat. Nos. 4,683,195,4,683,202, and 4,800,159.

[0164] According to a preferred embodiment, when the first-stageamplification is carried out, the 3′-end portion of the primer pair ofACP is involved in annealing at the first annealing temperature and whenthe second-stage amplification is carried out, the 5′-end portion of theprimer pair serves as a priming site. Such alteration of the portion toinvolve in annealing is mainly ascribed to the ACP itself, inparticular, the regulator portion of ACP. In the present methods, theregulator portion of ACP is capable of restricting the annealing portionof ACP to its 3′-end portion at the first annealing temperature,responsible for improving annealing specificity to a target sequence.

[0165] The present methods may be combined with many other processesknown in the art to achieve a specific aim. For example, the isolation(or purification) of amplified product may follow the second-stageamplification. This can be accomplished by gel electrophoresis, columnchromatography, affinity chromatography or hybridization. In addition,the amplified product of this invention may be inserted into suitablevehicle for cloning. Furthermore, the amplified product of thisinvention may be expressed in suitable host harboring expression vector.In order to express the amplified product, one would prepare anexpression vector that carries the amplified product under the controlof, or operatively linked to a promter. The promoter is originated fromthe vector itselt or the end portion of the amplified product, which maycorrespond to 5′-end portion of the ACP. Many standard techniques areavailable to construct expression vectors containing the amplifiedproduct and transcriptional/translational/control sequences in order toachieve protein or peptide expression in a variety of host-expressionsystems. The promoter used for prokaryotic host includes, but notlimited to, pLλ promoter, trp promoter, lac promoter and T7 promoter.The promoter used for eukaryotic host includes, but not limited to,metallothionein promoter, adenovirus late promoter, vaccinia virus 7.5Kpromoter and the promoters derived from polyoma, adenovirus 2, simianvirus 40 and cytomegalo virus. Certain examples of prokaryotic hosts areE. coli, Bacillus subtilis, and other enterobacteriaceae such asSalmonella typhimurium, Serratia marcescens, and various Pseudomonasspecies. In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. In addition to mammalian cells, these include insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus); and plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, tobacco mosaicvirus) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing one or more coding sequences. The expressedpolypeptide from the amplified product may be generally purified with avariety of purposes in accordance with the method known in the art.

[0166] In another aspect of this invention, there is provided a kit fornucleic acid amplification of the instant invention describedpreviously, which comprises the annealing control primer or annealingcontrol primer set indicated above.

[0167] In still another aspect of this invention, there is provided akit for selective amplification of a target nucleic acid sequence fromDNA described previously, which comprises the annealing control primeror annealing control primer set indicated above.

[0168] In further aspect of this invention, there is provided a kit forselective amplification of a target nucleic acid sequence from mRNAdescribed previously, which comprises the annealing control primer orannealing control primer set indicated above.

[0169] According to one embodiment of this invention, these kits furthercomprises a primer or a primer pair each having a nucleotide sequencecorresponding to the 5′-end portion of the ACP; in case that the 5′-endportion comprises universal primer sequence, it is more preferred thatthe kit comprises the universal primers. The present kits may optionallyinclude the reagents required for performing PCR reactions such asbuffers, DNA polymerase, DNA polymerase cofactors, anddeoxyribonucleotide-5′-triphosphates. Optionally, the kits may alsoinclude various polynucleotide molecules, reverse transcriptase, variousbuffers and reagents, and antibodies that inhibit DNA polymeraseactivity. The kits may also include reagents necessary for performingpositive and negative control reactions. Optimal amounts of reagents tobe used in a given reaction can be readily determined by the skilledartisan having the benefit of the current disclosure. The kits,typically, are adapted to contain in separate packaging or compartmentsthe constituents afore-described.

[0170] II. Application to Multiplex DNA Amplification

[0171] This application using ACP of the subject invention can alsoprovide an improved method for amplifying more than one target sequenceusing more than one pair of primers in the same reaction. In general, itis extremely difficult to set up PCR conditions to amplify more than 10targets in parallel because an optimal PCR reaction is required toamplify even one specific locus without any unspecific by-products, sothat those researchers who have achieved multiplex PCR have had to workhard to optimize their systems. Since annealing needs to take place at asufficiently high temperature to allow the perfect DNA-DNA matches tooccur in the reaction, the ACP of the subject invention is ideal in theoptimization of multiplex DNA amplification due to its function ofimproving the specificity of amplification. “Multiplex PCR” as usedherein refers to the simultaneous amplification of multiplex DNA targetsin a single polymerase chain reaction (PCR) mixture.

[0172] In still further aspect of this invention, there is provided amethod for amplifying more than one target nucleotide sequencesimultaneously using more than one pair of primers in the same reaction,wherein the method comprises performing an amplification reaction usingprimers, characterized in that at least one primer is derived from anyone of ACP described above. Preferably, the primer according to thestructure of ACP is one having at its 3′end portion a hybridizingsequence substantially complementary to a region of the target nucleicacid sequence to hybridize therewith.

[0173] In a specific embodiment of this invention, there is provided themethod using two stage amplifications, which comprises:

[0174] (a) performing a first-stage amplification of more than onetarget nucleotide sequence at a first annealing temperature comprisingat least two cycles of primer annealing, primer extending anddenaturing, using the primer pairs of any one of ACP above in which its3′end portion each of the primer pairs has a hybridizing nucleotidesequence substantially complementary to a region of the target nucleicacid sequence to hybridize therewith, under conditions in which each ofeach primer pair anneals to its target nucleotide sequence, whereby theamplification products of target nucleotide sequences are generated; and

[0175] (b) performing a second-stage amplification of the amplificationproducts generated from step (a) at a second annealing temperature,which is high stringent conditions, comprising at least one cycle ofprimer annealing, primer extending and denaturing, using the same primerpairs as used in step (a) or primer pairs each comprising a pre-selectedarbitrary nucleotide sequence corresponding to each 5′-end portion ofthe primer pairs used in step (a), under conditions in which each ofeach primer pair anneals to the 3′- and 5′-end sequences of theamplification products generated from step (a), respectively, wherebythe amplification products are re-amplified in the same reaction.

[0176] Since this application using the ACP of this invention is carriedout in accordance with the present methods for amplification of nucleicacid sequence previously discussed, except for using more than onetarget nucleotide sequence and primer pairs, the common descriptionsbetween them are omitted in order to avoid the complexity of thisspecification leading to undue multiplicity.

[0177] For instance, tjie composition and structure of ACP used and theconditions for amplification, are common between this process and thepresent methods for amplification of nucleic acid sequence previouslydiscussed.

[0178] In a preferred embodiment, the amplified products from each oftarget nucleotide sequences are different in size for subsequentanalysis.

[0179] According to a preferred embodiment, the amplification productsof multiplex target nucleotide sequences may be analyzed through sizeseparation. The size separation comparison is performed using a varietyof method known in the art, such as electrophoresis through apolyacrylamide gel matrix or agarose gel matrix and nucleotidesequencing. The nucleotide sequencing may be rapidly carried out with anautomatic sequencer available from various manufacturer.

[0180] As exemplified in Example below, the ACP of this inventionpermits the final amplified products to be free from the backgroundproblems as well as non-specificity arising from the conventionalprimers used in multiplex nucleic acid amplification methods known inthe art.

[0181] The advantage of the multiplex amplification is that numerousdiseases or specific nucleotide sequence alterations (e.g., singlenucleotide polymorphism or point mutation) can be assayed in the samereaction.

[0182] The number of analyses that can be run simultaneously isunlimited; however, the upper limit is probably about 20 and is likelyto be dependent on the size difference required for resolution andmethods that are available to resolve the amplified product.

[0183] In another aspect of this invention, there is provided a kit foramplifying more than one target nucleotide sequence simultaneously inthe same reaction, which comprises the annealing control primer orannealing control primer set described above. According to oneembodiment of this invention, these kits further comprises a primer or aprimer pair having a nucleotide sequence corresponding to the 5′-endportion of the ACP; in case that the 5′-end portion comprises universalprimer sequence, it is more preferred that the kit comprises theuniversal primers. The present kits may optionally include the reagentsrequired for performing PCR reactions such as buffers, DNA polymerase,DNA polymerase cofactors, and deoxyribonucleotide-5′-triphosphates.Optionally, the kits may also include various polynucleotide molecules,reverse transcriptase, various buffers and reagents, and antibodies thatinhibit DNA polymerase activity. The kits may also include reagentsnecessary for performing positive and negative control reactions.Optimal amounts of reagents to be used in a given reaction can bereadily determined by the skilled artisan having the benefit of thecurrent disclosure. The kits, typically, are adapted to contain inseparate packaging or compartments the constituents afore-described.

[0184] The method and kit of the present invention may be applied to thediagnosis of genetic and infectious diseases, gender determination,genetic linkage analysis, and forensic studies.

[0185] III. Application to Identification of Differentially ExpressedGenes

[0186] This application using ACP of the subject invention can alsoprovide an improved method for detecting and cloning cDNAs complementaryto differentially expressed mRNAs in two or more nucleic acid samples.

[0187] In still further aspect of this invention, there is provided amethod for detecting DNA complementary to differentially expressed mRNAin two or more nucleic acid samples, wherein the method comprisesreverse transcribing the mRNA and performing an amplification reactionusing primers, characterized in that at least one primer is derived fromany one of ACP described above. Preferably, the primer according to thestructure of ACP is one having at its 3′end portion a hybridizingsequence (more preferably, arbitrary sequence) substantiallycomplementary to a region of cDNA strands generated from reversetranscription.

[0188] In a specific embodiment of this invention, there is provided themethod using two stage amplifications, which comprises:

[0189] (a) providing a first sample of nucleic acids representing afirst population of mRNA transcripts and a second sample of nucleicacids representing a second population of mRNA transcripts;

[0190] (b) separately contacting each of the first nucleic acid sampleand the second nucleic acid sample with a first primer of any one of ACPdescribed above, in which the 3′-end portion of the first primercomprises a hybridizing nucleotide sequence substantially complementaryto a first site in the differentially expressed mRNA to hybridizetherewith, under conditions sufficient for template driven enzymaticdeoxyribonucleic acid synthesis to occur;

[0191] (c) reverse transcribing the differentially expressed mRNA towhich the first primer hybridizes to produce a first population of firstcDNA strands that are complementary to the differentially expressed mRNAin the first nucleic acid sample to which the first primer hybridizes,and a second population of first cDNA strands that are complementary tothe differentially expressed mRNA in the second nucleic acid sample towhich the first primer hybridizes;

[0192] (d) purifying and quantifying each of the first and secondpopulations of first cDNA strands;

[0193] (e) performing a first-stage amplification of each of the firstand second population of first DNA strands obtained from step (d) at afirst annealing temperature comprising at least one cycle of primerannealing, primer extending and denaturing, using a second primer of anyone of ACP described above having at its 3′ end portion a hybridizingsequence substantially complementary to a second site in the first andsecond populations of first cDNA strands, under conditions in which thesecond primer anneals to the second site in each population of the firstcDNA strands, whereby first and second populations of second cDNAstrands are generated;

[0194] (f) performing a second-stage amplification of each second cDNAstrand generated from step (e) at a second annealing temperature, whichis high stringent conditions, comprising at least two cycles of primerannealing, primer extending and denaturing, using the same first andsecond primers as used in steps (b) and (e), respectively, or a primerpair each comprising a pre-selected arbitrary nucleotide sequencecorresponding to each 5′-end portion of the first and second primersused in steps (b) and (e), respectively, under conditions in which eachprimer anneals to the 3′- and 5′-end sequences of each second cDNAstrand, respectively, whereby amplification products of the second cDNAstrands are generated, and

[0195] (g) comparing the presence or level of individual amplificationproducts in the first and second populations of amplification productsobtained from step (f).

[0196] Since this application using the ACP of this invention employsthe present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity.

[0197] A schematic representation for identifying differentiallyexpressed genes using novel ACP is illustrated in FIG. 2A.

[0198] In the present method, the nucleic acid sample representing apopulation of mRNA transcripts can be obtained from a wide variety ofbiological materials. In general, the first nucleic acid samplecomprises mRNA expressed in a first cell and the second nucleic acidsample comprises mRNA expressed in a second cell. In particular, thefirst nucleic acid sample comprises mRNA expressed in a cell at a firstdevelopmental stage and the second nucleic acid sample comprises mRNAexpressed in a cell at a second developmental stage. In addition, thefirst nucleic acid sample comprises mRNA expressed in a tumorigenic celland the second nucleic acid sample comprises mRNA expressed in a normalcell.

[0199] Steps (e) and (f) of the subject application may occur in asingle tube using the same reaction mixture except for primers, whichmeans that steps (e) and (f) are separated only in time. It would beunderstood that the primers corresponding to the 5′-end portion could beadded to the reaction mixture at the time of or after the second cDNAstrand synthesis. In a preferred embodiment, the primers correspondingto the 5′-end portion are added to the reaction mixture right after step(e) is completed, followed by subsequent PCR amplification of secondcDNA strands.

[0200] It would be also understood that the 5′-end portion sequences ofthe first and second ACPs used in steps (b) and (e), respectively, couldbe identical or different sequences; if they are identical, one primercorresponding to the sequence of 5′-end portion will be used in the step(f), whereas if they are different, two primers each corresponding tothe sequence of each 5′-end portion of ACPs will be used in the step(f). In a preferred embodiment, the 5′-end portion sequences of thefirst and second ACPs used in steps (b) and (e) are different and thus,two primers each corresponding to the sequence of each 5′-end portion ofACPs are used in step (f).

[0201] As an alternative process, in step (f) the complete sequences ofthe first and second ACPs used in steps (b) and (e), respectively,instead of the primers corresponding to the 5′-end portions of the ACPs,can be used as primers at the high stringent conditions for amplifyingeach second DNA strand obtained from step (e), wherein the 3′- and5′-ends of the second DNA strands which are initially synthesized usingthe second ACP comprise the sequence of the first ACP and thecomplementary sequence of the second ACP, respectively, and also serveas perfect pairing sites to the first and second ACPs. In this view,this alternative process is preferred because there is no need to addthe primers corresponding to the 5′-end portions of the ACPs to thereaction mixture at the time of or after first-stage PCR reaction. FIG.2B illustrates a schematic representation for identifying differentiallyexpressed genes by the alternative process stated above.

[0202] The method of the subject application for detecting differencesin gene expression uses only a single cDNA synthesis primer (the firstACP) to react with mRNA, unlike conventional Differential Display PCRwhich requires multiple cDNA synthesis anchor primers. In the originaldifferential display method outlined by Liang and Pardee in 1992, twelveanchor primers have been introduced. The anchor primers for example,having a sequence of T_(12 MN), where M is A, C, or G and N is A, C, Gor T, produced twelve separate cDNA populations. Recently, modifiedanchor primers have been proposed by altering the number of nucleotidessuch as one or three instead of two at the 3′-end which can hybridize toa sequence that is immediately 5′ to the poly A tail of mRNAs or byextending additional nucleotides at the 5′-end while retaining the Oligo(dT)_(9-12 MN) tail resulting in at least 21 nucleotides in length(Villeponteau et al., 1996, Combates et al., 2000).

[0203] The subject invention concerns the embodiments of the ACP used inthis method for the identification of differentially expressed genes,wherein the first ACP used in step (b) is represented by the followinggeneral formula (2): 5′-dX_(p)-dY_(q)-dT_(r)-3′ wherein dX is one of thefour deoxyribonucleotides, A, C, G, or T; dY is a regulator portioncomprising universal bases responsible for the main function of the ACPassociated with alteration of annealing temperature during PCR; dT is aT deoxyribonucleotide; p, q, and r represent an integer, respectively;dX_(p) represents the 5′-end portion and contains a pre-selectedarbitrary nucleotide sequence; dY_(q) contains at least 2 universalbases; dT_(r) represents the 3′-end portion; the nucleotide sequence ofthe 3′-end portion should have lower T_(m) than that of the 5′-endportion. The formula (2) basically follows the rule of formula (1). The3 ′-end portion of formula (2) consists of the sequences capable ofannealing to the poly A tail of mRNA and serves as a cDNA synthesisprimer for reverse transcription of mRNA.

[0204] In a preferred embodiment, the 3′-end portion of the first ACPused in step (b) contains at least 6 T nucleotides in length, which isconsidered a minimal requirement of length for primer annealing. Morepreferably, the 3′-end portion sequence is from 10 to 20 T nucleotidesand can be up to 30 T nucleotides in length. Most preferably, the 3′-endportion sequence is about 15 T nucleotides in length. This primer isnamed dT₁₅ annealing control primer (dT₁₅-ACP). In a preferredembodiment, the first primer has a general formula of5′-dX₁₅₋₃₀-dY₂₋₁₀-dT₁₀₋₂₀-3′, wherein dX represents adeoxyribonucleotide and comprises a pre-selected arbitrary nucleotidesequence not substantially complementary to the first and secondpopulations of mRNAs; dY represents the regulator portion comprising2-10 universal bases or non-discriminatory base analogs; and dTrepresents a contiguous deoxythymidine capable of annealing to the firstsite in the first and second populations of mRNAs.

[0205] In one embodiment, the 3′-end portion of the first ACP used instep (b) may contain at least one additional nucleotide at the 3′-endthat can hybridize to an mRNA sequence which is immediately upstream ofthe polyA tail. The additional nucleotides at the 3′ end of the firstACP may be up to 3 in length. For example, dT may further comprise 3′-Vat its 3′-end; in which V is one selected from the group consisting ofdeoxyadenosine, deoxycytidine and deoxyguanosine. In addition, dT mayfurther comprise 3′-NV at its 3′-end; in which V is one selected fromthe group consisting of deoxyadenosine, deoxycytidine and deoxyguanosineand N is one selected from the group consisting of deoxyadenosine,deoxythymidine, deoxycytidine and deoxyguanosine. Most preferably, the3′-end portion sequence of the first ACP used in step (b) contains dT₁₅only.

[0206] In a preferred embodiment, the first entire ACP is about 40-45nucleotides in length and comprises dT₁₅ at the 3′-end portion, dX₂₀₋₂₅at the 5′-end portion and dY₅ between the 3′- and 5′-end portions. Thefirst entire ACP can be up to 100 nucleotides in length. The firstprimer is exemplified by SEQ ID NOs: 30, 39, 57 and 61-63.

[0207] The first ACP described herein is hybridized to the poly A tailof the mRNA, which is present on all mRNAs, except for a small minorityof mRNA. The use of the first ACP used in this invention results in onlyone reaction and produces only one cDNA population, in contrast to atleast 3 to 64 separate cDNA populations generated by the conventionalanchor primers of Differential Display technique. This greatly increasesthe efficiency of the method by generating a substantially standard poolof single-stranded cDNA from each experimental mRNA population.

[0208] In the step (d), the standard pools of cDNAs synthesized by thefirst ACP should be purified and then quantitated by techniques wellknown to those of ordinary skill in the art such as spectrophotometry.This step is necessary to precisely control their inputs into theamplification step and then compare the final amplified poducts betweentwo or more samples Preferably, the amount of cDNA produced at thispoint in the method is measured. It is more preferred that thisdetermination is made using ultraviolet spectroscopy, although anystandard procedure known for quantifying cDNA known to those of ordinaryskill in the art is acceptable for use for this purpose. When using theUV spectroscopy procedure, an absorbance of about 260 nm of UV lightadvantageously is used. By the measurement of cDNA quantity at thisstep, therefore, the cDNA quantity can be standardized between or amongsamples in the following amplification reaction.

[0209] After synthesis of the first cDNA strands using the first ACP,the second cDNA strands are synthesized using the second ACP primerunder low stringent conditions, by at least one cycle comprisingdenaturing, annealing and primer extension, wherein the resultant firstcDNA strands are used as templates.

[0210] The second ACP basically follows the rule of formula (1) and its3′-end portion comprises a short arbitrary sequence, which preferablyhas lower T_(m) than that of the 5′-end portion. This primer is named anarbitrary annealing control primer (AR-ACP). In a preferred embodiment,the 3′-end portion of the second ACP can have from 8 to 15 nucleotidesin length. Most preferably, the 3′-end portion of the second ACPcontains about 10 nucleotides in length.

[0211] According to a preferred embodiment, the second ACP has thegeneral formula of 5′-dX₁₅₋₃₀-dY₂₋₁₀-dZ₈₋₁₅-3′, wherein dX represents adeoxyribonucleotide and comprises a pre-selected arbitrary nucleotidesequence not substantially complementary to the first and secondpopulations of the first cDNA strands; dY represents the regulatorportion comprising 2-10 universal bases or non-discriminatory baseanalogs; dZ represents a hybridizing arbitrary nucleotide sequencecapable of annealing to the second site in the first and secondpopulations of DNA strands. More preferably, the entire second ACP isabout 40-45 nucleotides in length comprising dZ₁₀ at the 3′-end portion,dX₂₀₋₂₅ at the 5′-end portion and dY₅ between the 3′- and 5′-endportions. The second entire ACP can be up to 100 nucleotides in length.The second primer is exemplified by SEQ ID NOs: 1-9, 13-18 and 20-23.

[0212] The second ACP described herein is different from a so-calledlong arbitrary primer, as used in the known modified DifferentialDisplay technique. For example, the conventional long arbitrary primersas described by Villeponteau et al. (1996) and Diachenko et al. (1996),having at least 21 or 25 nucleotides in length, comprise of onlyarbitrary nucleotides in the entire sequences. These conventional longarbitrary primers will hybridize in a non-predictable way under the lowannealing temperature (about 40° C.) which is required to achievearbitrary priming in the early PCR cycle, such that it is impossible todesign a representative set of primers rationally. Furthermore, many ofthe bands represent the same mRNA due to the “Stickiness” of longprimers when used under such a low stringency.

[0213] The advantages of the present method for detecting differentiallyexpressed genes are predominantly ascribed to the use of the second ACP.Since the second ACP is designed to limit the annealing of the secondACP to its 3′-end portion sequence, not to its 5′-end portion sequence,in association with annealing temperature, the resultant annealing willcome out in a predictable way, such that it is possible to design arepresentative set of primers rationally. In addition, the use of thesecond ACP allows avoiding false positive problems caused by the“Stickiness” of the conventional long primers under low stringentconditions as used in the previous Differential Display technique.

[0214] The annealing temperature used for the synthesis of second DNAstrands under low stringency conditions used in step (e) is preferablyabout between 40° C. to 65° C., more preferably, about between 45° C.and 55° C. and the most preferably, about 50° C. However, unlikeDifferential Display, which uses annealing temperatures between 35° C.and 45° C., the annealing temperature of low stringency conditions usedin the subject application is relatively higher than those used in theknown classical or enhanced Differential Display techniques witharbitrary primers.

[0215] Another unique and significant features of the subjectapplication for detecting differentially expressed genes is to amplifyonly the initially synthesized second DNA strands by the subsequentamplification, wherein the 3′- and 5′-ends of the second DNA strandswhich have been initially synthesized using the second ACP comprise thecomplementary sequence of the first ACP and the sequence of the secondACP, respectively and thus, the entire sequences of the first and secondACPs, or only their 5′-end portion sequences of the first and secondACPs, are used as 3′ and 5′ primer sequences for the amplification ofthe second DNA strands.

[0216] Since the ACP in the subject application leads to theamplification of specific products, it can be possible to fundamentallyeliminate the cause of major bottleneck problems, such as false productsand poor reproducibility, which result from non-specific annealing ofthe conventional arbitrary and dT primers to first and second DNAstrands as well as to amplified products during PCR in the knownDifferential Display methods.

[0217] In a preferred embodiment, the synthesis of second DNA strands instep (e) is carried out by at least 1 cycle of amplification under lowstringent conditions to achieve arbitrary priming, and through thesubsequent cycles, the amplification is processed more effectively forthe amplification of the resultant second DNA strands under highstringent conditions used in step (f). Most preferably, the synthesis ofsecond DNA strands in step (e) is carried out by one cycle ofamplification under low stringent conditions.

[0218] In a preferred embodiment, the amplification of the resultantsecond DNA strands synthesized by the step (e) is carried out under highstringent conditions using the complete sequences of the first andsecond ACPs used in steps (b) and (e), respectively, as primersequences, wherein the 3′- and 5′-ends of the resultant second DNAstrands provide perfect pairing sites to the first and second ACPs.However, it is interesting that the first and second ACPs are notinvolved in any other annealing to the template nucleic acid, except theannealing and extension of the 3′- and 5′- ends of the second DNAstrands as a reaction unit at such a high stringent condition becausetheir 3′-end portions require relatively low annealing temperature andthe high stringent conditions do not allow them to anneal to any site ofthe template, except the 3′- and 5′-ends of the second DNA strands.Consequently, owing to this function of ACP, which is capable ofselectively annealing to the template in associated with annealingtemperature, the amplified products can be free from the problems of thehigh false positive rate, poor reproducibility and possibleunder-representation of minor mRNA fractions in the analysis which arethe main problems of the known Differential Display. In this view, thereis a significant difference between this subject method and theconventional Differential Display methods despite the fact that they arein common to use the same primers for high stringent conditions as wellas for low stringent conditions.

[0219] In a preferred embodiment, the annealing temperature of theamplification for high stringent conditions used in step (f) ispreferably about between 55° C. and 72° C. Most preferably, theannealing temperature used for the high stringent conditions is about65-68° C.

[0220] In a preferred embodiment, the amplification under high stringentconditions used step (f) is carried out by at least 10 cycles and up to50 cycles to amplify the resultant second DNA strands synthesized bystep (e) during PCR. Most preferably, the PCR amplification is carriedout by 40-45 cycles.

[0221] The second-strand cDNA is preferably synthesized by PCR, morepreferably, hot start PCR method in which the procedure is to set up thecomplete reactions without the DNA polymerase and incubate the tubes inthe thermal cycler to complete the initial denaturation step at >90° C.Then, while holding the tubes at a temperature above 70° C., theappropriate amount of DNA polymerase can be pipetted into the reaction.In a preferred embodiment, the addition of the primers for thesecond-stage amplification into the reaction mixture after the completereaction of the second-strand cDNA synthesis is also carried out underdenaturation temperature such as >90° C. Then, while holding the tubesat a temperature about 90° C., the appropriate amount of the primers forthe second-stage amplification can be pipetted into the reaction.

[0222] An example of the second DNA strand synthesis and the subsequentamplification of the resultant second DNA strands in a single tube usingthe pre-selected arbitrary sequence of the 5′-end portions of the firstand second ACPs is conducted under the following conditions: the secondDNA strands are synthesized under low stringent conditions by one cycleof the first-stage amplification comprising annealing, extending anddenaturing reaction; the reaction mixture containing the first-strandcDNA, PCR reaction buffer (e.g., available from Roche), dNTP, and thesecond ACP is pre-heated at about 94° C., while holding the tubecontaining the reaction mixture at about 94° C., Taq polymerase (e.g.,available from Roche) is added into the reaction mixture; the PCRreactions are as follows: one cycle of 94° C. for 1 min, 50° C. for 3min, and 72° C. for 1 min; followed by denaturing the amplificationproduct at 94° C.; after the complete reaction of the second DNA strandsynthesis in step (e), 5′ pre-selected arbitrary primer and 3′pre-selected arbitrary primer are added to the reaction mixture and thenthe second stage amplification is conducted as follows: 40 cycles of 94°C. for 40 sec, 68° C. for 40 sec, and 72° C. for 40 sec; followed by a 5min final extension at 72° C.

[0223] An alternative example of the second DNA strand synthesis and thesubsequent amplification of the resultant second DNA strands in a singletube using the complete sequences of the first and second ACPs used insteps (b) and (e), respectively, instead of the pre-selected arbitrarysequences of the 5′-end portions of the first and second ACPs, isconducted under the following conditions: the second DNA strands aresynthesized under low stringent conditions by one cycle of thefirst-stage amplification comprising annealing, extending and denaturingreaction; the reaction mixture containing the first-strand cDNA, PCRreaction buffer (e.g., available from Roche), dNTP, the first ACP(dT₁₅-ACP), and the second ACP (AR-ACP) is pre-heated at about 94° C.,while holding the tube containing the reaction mixture at about 94° C.,Taq polymerase (e.g., available from Roche) is added into the reactionmixture; the PCR reactions are as follows: one cycle of 94° C. for 1min, 50° C. for 3 min, and 72° C. for 1 min; followed by thesecond-stage PCR amplification comprising annealing, extending anddenaturing reaction; the PCR reactions are as follows: 40 cycles of 94°C. for 40 sec, 65° C. for 40 sec, and 72° C. for 40 sec; followedc by a5 min final extension at 72° C.

[0224] It should be noted that a proper concentration of arbitrary ACP(the second ACP) is used to synthesize the second-strand cDNAs by onecycle of the first-stage amplification. If the amount of the second ACPused in the step (e) is too low, the resultant amplified products arenot reproducible. In contrast, the excess amount of the second ACP usedin the step (e) generates backgrounds such as DNA smear during PCR. In apreferred embodiment, the concentration of the second ACP used in thestep (e) is about between 0.1 μM and 1.0 μM. Most preferably, theconcentration of the second ACP as well as the first ACP is about 0.2μM. In a preferred embodiment, the concentration of the primers used inthe step (f) is about between 0.1 μM and 1 μM, most preferably, about0.4 μM.

[0225] Another significant feature of the subject application to theidentification of differences in gene expression is the use of highannealing temperature in a method. High annealing temperature used instep (f) increases the specificity of primer annealing during PCR, whichresults in eliminating false positive products completely and increasingreproducibility. Freedom from false positives which is one majorbottleneck remaining for the previous Differential Display technique isespecially important in the screening step for the verification of thecDNA fragments identified by Differential Display.

[0226] The step of comparing the presence or level of amplificationproducts obtained from step (f) may be performed in accordance withvarious methods known in the art. In a preferred embodiment, each of thefirst and second populations of amplification products of step (f) areresolved by electrophoresis to identify differentially expressed mRNAs.More preferably, the resultant PCR cDNA fragments are detected on anethidium bromide-stained agarose gel. Another prominent feature of thissubject application is the use of ethidium bromide-stained agarose gelto identify differentially expressed mRNAs. In general, the conventionalDifferential Display methods use radioactive detection techniques usingdenaturing polyacrylamide gels. However, according to the presentmethod, the significant amount of the amplified cDNA fragments obtainedthrough two stage amplifications allows to use an ethidiumbromide-stained agarose gel to detect the amplified cDNAs, which resultsin increasing the speed and avoiding the use of radioactivity.

[0227] Alternatively, the resulting cDNA fragments can be also detectedon a denaturing polyacrylamide gel by autoradiography or non-radioactivedetection methods such as silver staining (Gottschlich et al., 1997;Kociok et al., 1998), the use of fluoresenscent-labelledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), and the use of biotinylated primers (Korn etal., 1992; Tagle et al., 1993; Rosok et al., 1996).

[0228] In another embodiment, it might be useful for diagnostic purposesto use an automatic system such as an automatic DNA sequencer togetherwith any distinct labeling of the ACPs to detect or analyze theamplified products (Bauer, et al., 1993).

[0229] Considering the features of ACP in this subject application, thepresent method for detecting and cloning differentially expressed genesdiffers fundamentally from the previous Differential Display techniquesas described above.

[0230] In conclusion, the use of the ACP in this method makes itpossible to allow the amplification of only second DNA strands and theuse of the sufficient amount of starting materials as well as the highconcentration of dNTP, resulting in the following benefits: a)increasing primer annealing specificity, b) eliminating the problem offalse positives which requires the subsequent labor-intensive work toverify true positives, c) improving reliability and reproducibility, d)detecting rare mRNAs, e) generating long-distance PCR products rangingin size from 150 bp to 2.0 kb, f) allowing the use of ethidiumbromide-stained agarose gel to detect products, g) increasing the speedof analysis, h) particularly, not requiring well-trained hands toconduct this method, and i) allowing the rational design of arepresentative set of primers.

[0231] In further aspect of this invention, there is provided a kit fordetecting DNA complementary to differentially expressed mRNA, whichcomprises the annealing control primer or annealing control primer setdescribed above (the first and second primer). According to oneembodiment of this invention, these kits further comprises a primer or aprimer pair having a nucleotide sequence corresponding to the 5′-endportion of the ACPs; in case that the 5′-end portion comprises universalprimer sequence, it is more preferred that the kit comprises theuniversal primers. The present kits may optionally include the reagentsrequired for performing PCR reactions such as buffers, DNA polymerase,DNA polymerase cofactors, and deoxyribonucleotide-5′-triphosphates.Optionally, the kits may also include various polynucleotide molecules,reverse transcriptase, various buffers and reagents, and antibodies thatinhibit DNA polymerase activity. The kits may also include reagentsnecessary for performing positive and negative control reactions.Optimal amounts of reagents to be used in a given reaction can bereadily determined by the skilled artisan having the benefit of thecurrent disclosure. The kits, typically, are adapted to contain inseparate packaging or compartments the constituents afore-described.

[0232] IV. Application to Rapid Amplification of cDNA Ends (RACE)

[0233] This application using the ACP of the subject invention canprovide an improved method for rapidly amplifying cDNA ends, so calledRACE technologies. To be specific, the ACP of the subject application isadapted to the RACE technologies related to either of 3 ′- and 5′-end,and eliminates the background problems resulting from the primers usedin the conventional RACE technologies.

[0234] In still further aspect of this invention, there is provided amethod for rapidly amplifying a target cDNA fragment comprising a cDNAregion corresponding to the 3′-end region of an mRNA, wherein the methodcomprises reverse transcribing said mRNA and performing an amplificationreaction using primers, characterized in that at least one primer isderived from any one of ACPs described above. Preferably, the primeraccording to the structure of ACP is one having at its 3′-end portion agene-specific hybridizing nucleotide sequence substantiallycomplementary to a site in cDNA generated from reverse transcriptionand/or one having at its 3′-end portion a hybridizing nucleotidesequence substantially complementary to poly A tails of the mRNAs.

[0235] In a specific embodiment of this invention, there is provided themethod using two stage amplifications, which comprises:

[0236] (a) contacting mRNAs with a first primer of any one of the ACPdescribed above, in which the 3′-end portion of the primer comprises ahybridizing nucleotide sequence substantially complementary to poly Atails of the mRNAs to hybridize therewith, under conditions sufficientfor template driven enzymatic deoxyribonucleic acid synthesis to occur;

[0237] (b) reverse transcribing the mRNAs to which the first primerhybridizes to produce a population of first cDNA strands that arecomplementary to the mRNAs to which the first primer hybridizes;

[0238] (c) performing a first-stage amplification of the first cDNAstrands at a first annealing temperature comprising at least one cycleof primer annealing, primer extending and denaturing, using a secondprimer of any one of the ACP described above having at its 3′-endportion a gene-specific hybridizing nucleotide sequence substantiallycomplementary to a site in one of the first cDNA strands to hybridizetherewith, under conditions in which the second primer anneals to agene-specific site on one of the first cDNA strands, whereby agene-specific second cDNA strand is generated; and

[0239] (d) performing a second-stage amplification of the gene-specificsecond cDNA strand generated from step (c) at a second annealingtemperature, which is high stringent conditions, comprising at least twocycles of primer annealing, primer extending and denaturing, using thesame first and second primers as used in steps (a) and (c),respectively, or a primer pair each comprising a pre-selected arbitrarynucleotide sequence corresponding to each 5′-end portion of the firstand second primers used in steps (a) and (c), respectively, underconditions in which each primer anneals to the 3′- and 5′-end sequencesof a gene-specific second cDNA strand, respectively, whereby anamplification product of a gene-specific cDNA strand is generated.

[0240] Since this application using the ACP of this invention employsthe present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity.

[0241] A schematic representation for amplifying a target cDNA fragmentcomprising 3′-end region corresponding to the 3 -end of mRNA using novelACP system, called as ACP-based 3′ RACE, is illustrated in FIG. 3.

[0242] Steps (c) and (d) of the subject application may occur in asingle tube using the same reaction mixture except for primers, whichmeans that steps (c) and (d) are separated only in time. It would beunderstood that the primers corresponding to the 5′-end portion could beadded to the reaction mixture at the time of or after second cDNA strandsynthesis. In a preferred embodiment, the primers corresponding to the5′-end portion are added to the reaction mixture right after step (2) iscompleted, followed by subsequent amplification of second cDNA strands.

[0243] As an alternative process, in step (d) the complete sequences ofthe first and second ACPs, instead of the primers corresponding to the5′-end portions of the first and second ACPs, can be used as 3′ and 5′primers for amplifying the second-strand cDNA obtained from step (c),wherein the 3′- and 5′-ends of the second-strand cDNA which areinitially synthesized using the second ACP comprise the complementarysequence of the first ACP and the sequence of the second ACP,respectively, and also serve as perfect pairing sites to the first andsecond ACPs. FIG. 3 also illustrates a schematic representation foramplifying a target cDNA fragment comprising 3′-end region correspondingto the 3′-end of mRNA by the alternative process stated above.

[0244] One of significant features of the present invention for 3′-RACEis that the first ACP comprising nucleotide sequence substantiallycomplementary to poly A tail of mRNA is used as a cDNA synthesis primerand then the resultant cDNAs are directly used as templates forsubsequent amplification without any additional purification steps toremove the cDNA synthesis primer.

[0245] The annealing of the first ACP to the templates will beinterrupted during subsequent by the effect of the regulator portion onthe 3′- and 5′-end portions of the ACP under relatively high stringentconditions as described in the principle of ACP. As a result, thesubject application to 3′-RACE simplifies the conventional RACE methodsby reducing the step of purification and also, the ACP used in thesubject application does not involve the background problems because theannealing of the 3′-end portion is specified by the presence of theregulator portion positioned between the 3′-and 5′-end portions in theACPs, whereas the conventional cDNA synthesis primers such as Oligo-dTprimers for 3′-RACE generate backgrounds during PCR, which isnon-specific products. According to a preferred embodiment, the formulaof the first ACP for the cDNA synthesis may be identical to the formula(2).

[0246] When a gene-specific primer is used as 5′ primer, the firstamplification of a target cDNA fragment containing a 3′-end sequence instep (c) is carried out in accordance with conventional PCR methods asknown in the art. The term “gene-specific” in reference sequence usedherein refers to a partial sequence of a specific gene or complementthereof that has been generally known or available to one skilled in theart. Therefore, the gene-specific primer means one comprising thegene-specific sequence.

[0247] The generated second cDNA strand is amplified by the second-stageamplification which is used in the application of the present inventionfor amplifying a target nucleic acid sequence above. Since the ACPdescribed in this invention can generate stable T_(m) in a primer andalso tolerate “primer search parameters” for primer design such asprimer length, annealing temperature, GC content, and PCR productlength, it is useful when the gene-specific primer sequences have lowT_(m) or are too sensitive to such parameters to generate specificproducts.

[0248] In another aspect of this invention, there is provided a methodfor rapidly amplifying a target DNA fragment comprising a cDNA regioncorresponding to the 5′-end region of an niRNA, wherein the methodcomprises reverse transcribing the mRNA and performing an amplificationreaction using primers, characterized in that at least one primer isderived from any one of ACPs described above. Preferably, the primeraccording to the structure of ACP is one having at its 3′-end portion agene-specific hybridizing nucleotide sequence substantiallycomplementary to a site in cDNA generated from reverse transcription.

[0249] In a specific embodiment of this invention, there is provided themethod using two stage amplifications, which comprises:

[0250] (a) contacting mRNAs with an oligonucleotide dT primer or randomprimer as a cDNA synthesis primer under conditions sufficient fortemplate driven enzymatic deoxyribonucleic acid synthesis to occur, inwhich the cDNA synthesis primer comprises a hybridizing nucleotidesequence substantially complementary to a region of an mRNA to hybridizetherewith;

[0251] (b) reverse transcribing the mRNAs, using a reversetranscriptase, to which the cDNA synthesis primer hybridizes to producea population of first cDNA strands that are complementary to the mRNAsto which the cDNA synthesis primer hybridizes, whereby mRNA-cDNAintermediates are generated;

[0252] (c) permitting cytosine residues to be tailed at the 3′-ends ofthe first cDNA strands in the form of the mRNA-cDNA intermediates by theterminal transferase reaction of reverse transcriptase;

[0253] (d) contacting the cytosine tails at the 3′-ends of the firstcDNA strands generated from step (c) with an oligonucleotide whichcomprises a 3′-end portion and a 5′-end portion separated by a group ofuniversal base or non-discriminatory base analog, wherein the 3′-endportion comprises at least three guanine residues at its 3′-end tohybridize with the cytosine tails at the 3′-ends of the first cDNAstrands and the 5′-end portion comprises a pre-selected arbitrarynucleotide sequence, under conditions in which the 3-end portion of theoligonucleotide is hybridized to the cytosine tails;

[0254] (e) extending the tailed 3′-ends of the first cDNA strands togenerate an additional 30 sequence complementary to the oligonucleotideusing reverse transcriptase, in which the oligonucleotide serves as atemplate in the extension reaction, whereby full-length first cDNAstrands are extended;

[0255] (f) performing a first-stage amplification of the full-lengthfirst cDNA strands obtained from step (e) at a first annealingtemperature comprising (i) and (ii) as follows:

[0256] (i) at least one cycle of primer annealing, primer extending anddenaturing using a first primer comprising a nucleotide sequencesubstantially complementary to the 3′-end sequences of the full-lengthfirst cDNA strands under conditions in which the first primer anneals tothe full-length first cDNA strands, under conditions in which the firstprimer anneals to the 3′- ends of the full-length first cDNA strands,whereby full-length second cDNA strands are generated;

[0257] (ii) at least one cycle of primer annealing, primer extending anddenaturing using a 10 second primer of any one of claims 1-25 having atits 3′-end portion a gene-specific hybridizing sequence substantiallycomplementary to a region on one of the full-length second cDNA strandsto hybridize therewith, under conditions in which the second primeranneals to a gene-specific site on one of the full-length second cDNAstrands, whereby a gene-specific cDNA strand is generated; and

[0258] (g) performing a second-stage amplification of the gene-specificcDNA strand at a second annealing temperature, which is high stringentconditions, comprising at least two cycles of primer annealing, primerextending and denaturing, using the same first and second primers asused in steps (f)-(i) and (f)-(ii), respectively, or a primer pair eachcomprising a nucleotide sequence corresponding to each 5′-end portion ofthe first and second primers as used in steps (f)-(i) and (i)-(ii),respectively, under conditions in which each primer anneals to the 3′-and 5′-end sequences of a gene-specific cDNA strand, respectively,whereby an amplification product of a gene-specific cDNA strand isgenerated.

[0259] The schematic representations for amplifying a target cDNAfragment comprising 5′-end region corresponding to the 5′-end of mRNAusing novel ACP system, called as ACP-based 5′ RACE, is illustrated inFIGS. 4A (using oligonucleotide dT primer) and 4B (using random primer).

[0260] The descriptions of the oligonucleotide dT primer used in step(a) is identical to those used in the present method for amplificationof a target nucleic acid sequence from an mRNA.

[0261] Alternatively, when the size of a target mRNA is so large thatthe reverse transcriptase falls off before reaching the 5′ completesequences, the random primer is used as cDNA synthesis primer.

[0262] According to a preferred embodiment, the step (c), permittingcytosine residues to be tailed is performed in the presence of manganeseion.

[0263] Steps (f) and (g) of the subject application may occur in asingle tube using the same reaction mixture except for primers, whichmeans that steps (f) and (g) are separated only in time. It would beunderstood that the primer(s) used in each step (f) and (g) can be addedto the reaction mixture at the time of or after each step. In apreferred embodiment, the primer(s) is(are) added to the reactionmixture right after each step is completed, followed by subsequent PCRamplification of second cDNA strands.

[0264] When a gene-specific primer is used as 5′ primer, theamplification of a target cDNA fragment containing a 5′-end sequence instep (f) is carried out under high stringent conditions in accordancewith conventional PCR methods as known in the art.

[0265] In a preferred embodiment, a target cDNA fragment containing a5′-end sequence in step (f) is amplified using a second ACP comprising agene-specific sequence at the 3′-end portion, by two stage PCRamplifications which are used in the application of the presentinvention for amplifying a target nucleic acid sequence above. Since theACP described in this invention can generate stable T_(m) in a primerand also tolerate “primer search parameters” such as primer design,comprising primer length, annealing temperature, GC content, and PCRproduct length, it is particularly useful when the gene-specific primersequences have low T_(m) or are too sensitive to such parameters togenerate specific products. The formula of the second ACP is identicalto the formula (1) in which the 3′-end portion contains a gene-specificsequence.

[0266] The oligonucleotide for the step (d) is similar to CapFinderprimer (Chenchik et al., 1998; Chenchik et al. U.S. Pat. Nos. 5,962,271and 5,962,272) in the senses both of them comprise at least threeguanine residues at its 3′-end and use them as a template switchingprimer for the 3′-end extension of the first cDNA strand by reversetranscriptase, whereas they are clearly different from each other interms of the function of a switch in controlling primer annealing to atemplate nucleic acid in associated with annealing temperature duringPCR. CapFinder primer does not comprise univeasl base residue groupwhich is responsible for regulating primer annealing in ACP, so that theCapFinder PCR method for 5′-RACE (Chenchik et al., 1998) can not be freefrom a high background such as DNA spear arising from contamination ofthe primers such as the CapFinder and Oligo-dT primers used in cDNAsynthesis during PCR. On the other hand, the universal base resiudegroup of the first ACP plays a key role in regulating primer annealing,so that the subject method does not provide any cause for the backgroundproblems during subsequent PCR amplification; this is a key feature ofthe ACP application to 5′-RACE.

[0267] Furthermore, when the ACP of the present invention is used in5′-RACE technology, it is unnecessary to conduct the process of physicalseparation such as a solid-phase cDNA synthesis and procedures which hasbeen introduced as an alternative method to remove all contaminants usedin cDNA synthesis (Schramm et al., 2000).

[0268] In a preferred embodiment, the oligonucleotide to form abase-pair(s) with the cytosine tail for 5′-RACE which has a similarstructure to ACP, wherein the oligonucleotide is represented by thefollowing general formula (3): 5′-dX₁₅₋₃₀dY₂₋₁₀-dZ₁₋₁₀-G₃₋₅-3′, in whichdX represents a deoxyribonucleotide and comprises a pre-selectedarbitrary sequence; dY represents a regulatory portion comprising 2-10universal bases or non-discriminatory base analogs; dZ represents adeoxyribonucleotide and comprises a pre-selected arbitrary sequence; andG₃₋₅ represents three to five guanines.

[0269] Most preferably, the 3′-end portion sequence dZ is about 2-3nucleotides in length. Further, in one embodiment, the 5′-end portion dXcan include a sequence that is recognized by a restriction endonuclease.

[0270] The G₃₋₅ may be three to five riboguanines or deoxyguanines, or acombination of riboguanine and deoxyriboguanine. In more preferredembodiment, the G₃₋₅ comprises two riboguanines and one deoxyriboguanine(r(G)₂-d(G)-3′), most preferably, three riboguanines.

[0271] When the gene-specific primer in step (f) is used as 3′ primerfor 5′-RACE, a target cDNA fragment containing a 5′-end sequence isamplified under high stringency conditions by conventional PCR methodsas known in the art.

[0272] In a preferred embodiment, a target cDNA fragment containing a5′-end sequence is amplified using a second ACP which comprises agene-specific sequence at the 3′-end portion, by two stage PCRamplifications which is conducted in the application for amplifying atarget nucleic acid sequence in the present invention. Since the ACPdescribed in this invention can provide stable T_(m) in a primer andalso tolerate “primer search parameters” for primer design such asprimer length, annealing temperature, GC content, and PCR productlength, it is useful when the gene-specific primer sequences have lowT_(m) or are too sensitive to such parameters to generate specificproducts. The formula of the second ACP is identical to the formula (1)in which the 3′-end portion contains a gene-specific sequence. p The useof ACP in RACE technology significantly simplifies and improves theconventional RACE technologies with regard to the amplification of cDNAends as described above. The vital feature of the subject method is tobe free from the background problems arising from the primers used inconventional RACE methods. Consequently this method described herein canbe more effective, easier, less labor-intensive, and more reproduciblethan conventional RACE methods.

[0273] In still another aspect of this invention, there is provided akit for rapidly amplifying a target cDNA fragment comprising 3′-endregion of mRNA, which comprises the annealing control primer orannealing control primer set described previously (including the firstand second primer). According to one embodiment of this invention, thesekits further comprises a primer or a primer pair having a nucleotidesequence corresponding to the 5′-end portion of the ACPs; in case thatthe 5′-end portion comprises universal primer sequence, it is morepreferred that the kit comprises the universal primers. The present kitsmay optionally include the reagents required for performing PCRreactions such as buffers, DNA polymerase, DNA polymerase cofactors, anddeoxyribonucleotide-5′-triphosphates. Optionally, the kits may alsoinclude various polynucleotide molecules, reverse transcriptase, variousbuffers and reagents, and antibodies that inhibit DNA polymeraseactivity. The kits may also include reagents necessary for performingpositive and negative control reactions. Optimal amounts of reagents tobe used in a given reaction can be readily determined by the skilledartisan having the benefit of the current disclosure. The kits,typically, are adapted to contain in separate packaging or compartmentsthe constituents afore-described.

[0274] In further aspect of this invention, there is provided a kit forrapidly amplifying a target cDNA fragment comprising 5′-end region ofmRNA, which comprises the annealing control primer or annealing controlprimer set described above (including the oligonucleotide dT primer andrandom primer for cDNA synthesis, the oligonucleotide to form abase-pair(s) with the cytosine tail, the first primer and the secondprimer). According to one embodiment of this invention, these kitsfurther comprises a primer pair each comprising a nucleotide sequencecorresponding to each 5′-end portion of the first and second primers asused in steps (f)-(i) and (f)-(ii); in case that the 5′-end portioncomprises universal primer sequence, it is more preferred that the kitcomprises the universal primers.

[0275] V. Application to Amplifying Full-length cDNA

[0276] In further aspect of this invention, there is provided a methodfor amplifying a population of full-length double-stranded cDNAscomplementary to mRNAs, wherein the method comprises reversetranscribing the mRNA and performing an amplification reaction usingprimers, characterized in that at least one primer is derived from anyone of ACP described above. Preferably, the primer having the structureof ACP is one having a hybridizing nucleotide sequence substantiallycomplementary to poly A tails of mRNAs.

[0277] In a specific embodiment of this invention, there is provided themethod comprises:

[0278] (a) contacting the mRNAs with a first primer of any one of ACPdescribed above, in which the 3′-end portion of the first primer has ahybridizing nucleotide sequence substantially complementary to poly Atails of the mRNAs to hybridize therewith, under conditions sufficientfor template driven enzymatic deoxyribonucleic acid synthesis to occur;

[0279] (b) reverse transcribing the mRNAs, using a reversetranscriptase, to which the first primer hybridizes to produce thepopulation of first cDNA strands that are complementary to the mRNAs towhich the primer hybridizes, whereby mRNA-cDNA intermediates aregenerated;

[0280] (c) permitting cytosine residues to be tailed at the 3′-ends ofthe first cDNA strands in the form of the mRNA-cDNA intermediates by theterminal transferase reaction of reverse transcriptase;

[0281] (d) contacting the cytosine tails at the 3′-ends of the firstcDNA strands generated from step (c) with an oligonucleotide whichcomprises a 3′-end portion and a 5′-end portion separated by a group ofuniversal base or non-discriminatory base analog, wherein the 3′-endportion comprises at least three guanine residues at its 3′-end tohybridize with the cytosine tails at the 3′-ends of the first cDNAstrands and the 5′-end portion comprises a pre-selected arbitrarynucleotide sequence, under conditions in which the 3-end portion of theoligonucleotide is hybridized to the cytosine tails;

[0282] (e) extending the tailed 3′-ends of the first cDNA strands togenerate an additional sequence complementary to the oligonucleotideusing reverse transcriptase, in which the oligonucleotide serves as atemplate in the extension reaction, whereby full-length first cDNAstrands are extended; and

[0283] (f) performing an amplification of the full-length first cDNAstrands generated from step (e) comprising at least two cycles of primerannealing, primer extending and denaturing, using a primer pair eachcomprising a nucleotide sequence corresponding to the same first primerand oligonucleotide as used in steps (a) and (d), respectively, or aprimer pair each comprising a nucleotide sequence corresponding to each5′-end portion of the first primer and oligonucleotide used in steps (a)and (d), respectively, under conditions in which each primer anneals tothe 3′- and 5′-end sequences of the full-length first cDNA strands,respectively, whereby amplification products of full-length cDNA strandscomplementary to the mRNAs are generated.

[0284] Since this application using the ACP of this invention employs inprinciple the present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity. In addition, the ACP described above in which the 3′-endportion has a hybridizing nucleotide sequence substantiallycomplementary to poly A tails is in principle identical to the firstprimer for the present method for 3′-RACE. Furthermore, theoligonucleotide to form a base-pair(s) with the cytosine tail and theprimer pair used in the step (f) are in principle identical to those for5′-RACE of this invention discussed above.

[0285] A schematic representation for amplifying full-length cDNAmolecules of the present invention is illustrated in FIG. 5.

[0286] The use of ACP significantly simplifies and improves theconventional technologies with regard to the amplification offull-length cDNAs as described above. The vital feature of the subjectmethod is to be free from the background problems arising from theprimers used in conventional methods. Consequently this method describedherein can be more effective, easier, less labor-intensive, and morereproducible than conventional methods.

[0287] In still further aspect of this invention, there is provided akit for amplifying a full-length double stranded cDNA complementary tomRNA, which comprises the annealing control primer or the annealingcontrol primer set described above (including the oligonucleotide dTprimer, the oligonucleotide to form a base-pair(s) with the cytosinetail, the primer(s) used in the step (f)). According to one embodimentof this invention, these kits further comprises a primer pair eachcomprising a nucleotide sequence corresponding to each 5′-end portion ofthe primer and oligonucleotide used in steps (a) and (d), respectively;in case that the 5′-end portion comprises universal primer sequence, itis more preferred that the kit comprises the universal primers. Thepresent kits may optionally include the reagents required for performingPCR reactions such as buffers, DNA polymerase, DNA polymerase cofactors,and deoxyribonucleotide-5′-triphosphates. Optionally, the kits may alsoinclude various polynucleotide molecules, reverse transcriptase, variousbuffers and reagents, and antibodies that inhibit DNA polymeraseactivity. The kits may also include reagents necessary for performingpositive and negative control reactions. Optimal amounts of reagents tobe used in a given reaction can be readily determined by the skilledartisan having the benefit of the current disclosure. The kits,typically, are adapted to contain in separate packaging or compartmentsthe constituents afore-described.

[0288] VI. Application to Amplifying 5′-Enriched cDNA

[0289] In another aspect of this invention, there is provided a methodfor amplifying a population of 5′ -enriched double-stranded cDNAscomprising cDNA regions corresponding to the 5′-end regions of mRNAs,wherein the method comprises reverse transcribing the mRNA andperforming an amplification reaction using primers, characterized inthat at least one primer is derived from any one of ACP described above.Preferably, the primer having the structure of ACP used for cDNAsynthesis is one having at its 3′-end portion at least six randomnucleotide sequences.

[0290] In a specific embodiment of this invention, there is provided themethod comprises:

[0291] (a) contacting the mRNAs with a first primer of any one of ACPdescribed above under conditions sufficient for template drivenenzymatic deoxyribonucleic acid synthesis to occur, wherein the 3′-endportion of the first primer has at least six random nucleotidesequences;

[0292] (b) performing the steps (b)-(e) of the method for amplifying apopulation of full-length double-stranded cDNAs, whereby 5′-enrichedfirst cDNA strands are extended;

[0293] (c) performing an amplification of the 5′-enriched first cDNAstrands generated from step (b) comprising at least two cycles of primerannealing, primer extending and denaturing, using a primer pair eachcomprising a nucleotide sequence corresponding to each 5′-end portion ofthe primer and oligonucleotide used in steps (a) and (b), respectively,under conditions in which each primer anneals to the 3′- and 5′-endsequences of the 5′-enriched first cDNA strands, respectively, wherebyamplification products of 5′-enriched cDNA strands are generated.

[0294] Since this application using the ACP of this invention employs inprinciple the present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity. In addition, the oligonucleotide to form a base-pair(s)with the cytosine tail and the primer pair used in the step (c) are inprinciple identical to those for 5′-RACE of this invention discussedabove. A schematic representation for the method for amplifying5′-enriched double-stranded cDNAs complementary to mRNAs is illustratedin FIG. 6.

[0295] “5′ enriched cDNAs” refers to a significant portion of the cDNAconstituents which contain the nucleotide sequence information of the5′-end of the mRNAs from which the cDNAs are derived.

[0296] The formula of the first primer is identical to the formula (1)in which the 3′-end portion comprises a random nucleotide sequence. In apreferred embodiment, the 3′-end portion of the first primer used instep (a) contains at least six random deoxyribonucleotides. In apreferred embodiment, the 5′-end portion of the first primer used instep (a) can includes a sequence that is recognized by a restrictionendonuclease. The conventional methods require more steps to amplify 5′enriched cDNA molecules complementary to the mRNA molecules than thesubject method because the conventional methods use the conventionalprimers which do not have the function of controlling primer annealing.In contrast, this subject method is considerably a simple and effectiveapproach due to the function of regulating primer annealing generated bythe effect of a universal base residue group in ACP.

[0297] In still another aspect of this invention, there is provided akit for amplifying 5′-enriched double-stranded cDNAs complementary tomRNAs, which comprises the annealing control primer or the annealingcontrol primer set described above (the first primer, theoligonucleotide to form a base-pair(s) with the cytosine tail).According to one embodiment of this invention, these kits furthercomprises a primer pair each comprising a nucleotide sequencecorresponding to each 5′-end portion of the primer and oligonucleotideused in steps (a) and (b), respectively;

[0298] in case that the 5′-end portion comprises universal primersequence, it is more preferred that the kit comprises the universalprimers. The present kits may optionally include the reagents requiredfor performing PCR reactions such as buffers, DNA polymerase, DNApolymerase cofactors, and deoxyribonucleotide-5′-triphosphates.Optionally, the kits may also include various polynucleotide molecules,reverse transcriptase, various buffers and reagents, and antibodies thatinhibit DNA polymerase activity. The kits may also include reagentsnecessary for performing positive and negative control reactions.Optimal amounts of reagents to be used in a given reaction can bereadily determined by the skilled artisan having the benefit of thecurrent disclosure. The kits, typically, are adapted to contain inseparate packaging or compartments the constituents afore-described.

[0299] VII. Application to DNA or RNA Fingerprinting

[0300] This application using ACP of the subject invention can providean improved method for detecting polymorphisms in genomic DNA (DNAfingerprinting) or for detecting differential gene expression in mRNA(RNA fingerprinting).

[0301] In further aspect of this invention, there is provided a methodfor producing a DNA fingerprint of gDNA, wherein the method comprisesperforming an amplification reaction using primers, characterized inthat at least one primer is derived from any one of ACPs describedabove. Preferably, the primer having the structure of ACP is one havingat its 3′-end portion an arbitrary nucleotide sequence substantiallycomplementary to sites on the gDNA.

[0302] In a specific embodiment of this invention, there is provided themethod using two stage amplifications, which comprises:

[0303] (a) performing a first-stage amplification of the DNAfingerprint, which is a set of discrete DNA segments characteristic ofgenome, from the gDNA at a first annealing temperature comprising atleast two cycles of primer annealing, primer extending and denaturing,using the primer or the primer pair of any one of ACPs described above,wherein each primer has at its 3′-end portion an arbitrary nucleotidesequence substantially complementary to sites on the gDNA to hybridizetherewith, under conditions in which the primer or the primer pairanneals to the gDNA, whereby the set of discrete DNA segmentscharacterized as a DNA fingerprint is produced; and

[0304] (b) performing a second-stage amplification of the set ofdiscrete DNA segments generated from step (a) at a second annealingtemperature, which is high stringent conditions, comprising at least onecycle of primer annealing, primer extending and denaturing, using thesame primer or primer pair as used in step (a) or a primer or a primerpair each comprising a nucleotide sequence corresponding to each 5′-endportion of the primer or primer pair used in step (a), under conditionsin which the primer or each of the primer pair anneals to the 3′- and5′-end sequences of the set of discrete DNA segments generated from step(a), respectively, whereby the set of discrete DNA segments isre-amplified.

[0305] In still further aspect of this invention, there is provided amethod for producing a RNA fingerprint of an mRNA sample, wherein themethod comprises reverse transcribing and performing an amplificationreaction using primers, characterized in that at least one primer isderived from any one of ACPs. Preferably, the primer according to thestructure of ACP is one having at its 3′-end portion an arbitrarynucleotide sequence substantially complementary to sites on cDNA strandsgenerated from reverse transcription and/or one having at its 3′-endportion a hybridizing nucleotide sequence substantially complementary topoly A tails of the mRNAs.

[0306] In a specific embodiment of this invention, there is provided themethod using two stage amplifications, which comprises:

[0307] (a) contacting the mRNA sample with a first primer of any one ofACPs described above, in which the first primer has a hybridizingnucleotide sequence substantially complementary to poly A tails of themRNA sample to hybridize therewith, under conditions sufficient fortemplate driven enzymatic deoxyribonucleic acid synthesis to occur;

[0308] (b) reverse transcribing the mRNA sample to which the firstprimer hybridizes to produce a population of first cDNA strands that arecomplementary to the mRNA sample to which the first primer hybridizes;

[0309] (c) performing a first-stage amplification of the population offirst cDNA strands generated from step (b) at a first annealingtemperature comprising at least one cycle of primer annealing, primerextending and denaturing, using a second primer or primer pair of anyone of ACPs described above, wherein each primer has at its 3′-endportion an arbitrary nucleotide sequence substantially complementary tosites on the first cDNA strands to hybridize therewith, under conditionsin which the primer or primer pair anneals to the mRNA sample, whereby aset of discrete cDNA segments characterized as a RNA fingerprint isproduced; and

[0310] (d) performing a second stage amplification of the set ofdiscrete cDNA segments generated from step (c) at a second annealingtemperature which is high stringent conditions, comprising at least onecycle of primer annealing, primer extending and denaturing, using thesame primer or primer pair as used in step (c) or a primer or primerpair each comprising a nucleotide sequence corresponding to each 5′-endportion of the primer or primer pair used in step (c), under conditionsin which the primer or each of the primer pair anneals to the 3′- and 5′-end sequences of the set of discrete cDNA segments generated from step(c), respectively, whereby the set of discrete cDNA segments isre-amplified.

[0311] Since this application using the ACP of this invention employs inprinciple the present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity. In addition, the RNA fingerprinting in principle followsthe present method for detecting DNA complementary to differentiallyexpressed mRNA.

[0312] The term “genomic DNA” as used herein refers to a population ofDNA that comprises the complete genetic component of a species. Thusgenomic DNA comprises the complete set of genes present in apre-selected species. The complete set of genes in a species is alsoreferred to as genome. The term DNA or RNA “fingerprinting” as usedherein refers to a set of discrete DNA amplification productscharacteristic of a genome or a set of discrete cDNA segmentscharacteristic of a sample of mRNA, respectively.

[0313] In the previous arbitrarily primed PCR fingerprints, calledAP-PCR, short or long arbitrary primers have been used undernon-stringent conditions for early 2-5 cycles of PCR amplificationbecause a low annealing temperature is required to achieve arbitrarypriming, such that a significant portion of isolated fragments is notstill reproducible although effective amplification proceeds in thefollowing cycles under high stringent condition.

[0314] In contrast to AP-PCR, the ACP-based PCR for fingerprintingincreases the specificity of primer annealing during PCR due to thefunction of a universal base residue group positioned between the 3′-and 5′-end portions of ACP, wherein the universal base residue grouprestricts the annealing site to the 3′-end portion of the ACP and alsoallows this 3′-end portion to anneal at a relatively high annealingtemperature. Thus, the ACP-based PCR for fingerprinting completelyeliminates false positive products and significantly increasesreproducibility.

[0315] In a preferred embodiment, the ACP contains an arbitrary sequenceat the 3′-end portion with at least 6 nucleotides in length. Morepreferably, the 3′-end portion contains 8-15 nucleotides in length, mostpreferably, about 10 nucleotides in length.

[0316] A single ACP or a pair of ACPs can be used for detectingpolymorphisms in DNA fingerprinting. Preferably, a pair of ACPs is usedfor DNA fingerprinting because a pair of ACPs produces more productsthan a single arbitrary ACP does.

[0317] An example of the DNA fingerprinting using ACP is conducted bytwo stages of PCR amplifications under the following conditions:amplification reactions are performed under low stringent conditions bytwo cycles of the first-stage PCR comprising annealing, extending anddenaturing reaction; the reaction mixture containing genomic DNA, PCRreaction buffer, MgCl₂, dNTPs (dATP, dCTP, dGTP and dTTP), a pair ofACPs is pre-heated, Taq polymerase is added into the reaction mixture;the PCR reactions are performed, followed by denaturing theamplification product; after the complete reaction of the first-stagePCR, the pre-selected arbitrary primer JYC4 corresponding to the 5′-endportion of the ACPs are added to the reaction mixture and then thesecond stage PCR amplification is conducted.

[0318] It should be noted that a proper concentration of ACP is used toproduce DNA fingerprinting. If the amount of the ACP is too low, theresultant amplified PCR products are not reproducible. In contrast, theexcess amount of the ACP generates backgrounds such as DNA smear duringPCR. In a preferred embodiment, the concentration of the ACP is aboutbetween 0.1 μM and 2 μM. Most preferably, the concentration of the ACPis about 1.4 μM.

[0319] In a preferred embodiment, the concentration of the primercorresponding to the 5′-end portion of the ACPs is about between 0.1 μMand 2 μM, most preferably, about 0.8 μM.

[0320] The genomic DNA and mRNA samples may be obtained from a widevariety of biomaterials and conditions. For example, they may beobtained from plants, animal (human) and microbes and from differentindividual organisms.

[0321] The amplified products can be analyzed by gel electrophoresis. Inone embodiment, the resulting PCR products can be also detected on adenaturing polyacrylamide gel by autoradiography or non-radioactivedetection methods such as silver staining (Gottschlich et al., 1997;Kociok et al., 1998), the use of fluoresenscent-labelledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), and the use of biotinylated primers (Korn etal., 1992; Tagle et al., 1993; Rosok et al., 1996).

[0322] In still further aspect of this invention, there is provided akit for producing a DNA fingerprint by use of gDNA or mRNA, whichcomprises the annealing control primer or annealing control primer setdescribed above. The descriptions of the kits for the amplification ofnucleic acid sequence and for detecting DNA complementary todifferentially expressed mRNA of this invention can be applied to thepresent kit.

[0323] VIII. Application to Identification of Conserved HomologySegments in Multigene Families

[0324] This application using ACP of the subject invention can alsoprovide an improved method for the identification of conserved homologysegments in multigene families.

[0325] In another aspect of this invention, there is provided a methodfor identifying conserved homology segments in a multigene family froman mRNA sample, wherein the method comprises reverse transcribing andperforming an amplification reaction using primers, characterized inthat at least one primer is derived from any one of ACPs describedabove. Preferably, the primer having the structure of ACP is one havingat its 3′-end portion a hybridizing sequence substantially complementaryto a consensus sequence or a degenerate sequence encoding amino acidsequence of a conserved homology segment on cDNA strands generated fromreverse transcription and/or one having at its 3′-end portion ahybridizing nucleotide sequence substantially complementary to poly Atails of the mRNAs.

[0326] In a specific embodiment of this invention, there is provided themethod using two stage amplifications, which comprises:

[0327] (a) contacting the mRNA sample with a first primer of any one ofclaims 1-29, in which the first primer has a hybridizing nucleotidesequence substantially complementary to poly A tails of the mRNA sampleto hybridize therewith, under conditions sufficient for template drivenenzymatic deoxyribonucleic acid synthesis to occur;

[0328] (b) reverse transcribing the mRNA sample to which the firstprimer hybridizes to produce a population of first cDNA strands that arecomplementary to the mRNA sample to which the first primer hybridizes;

[0329] (c) performing a first-stage amplification of the population offirst cDNA strands generated from step (b) at a first annealingtemperature comprising at least one cycle of primer annealing, primerextending and denaturing, using a second primer of any one of claims1-25 having at its 3′ end portion a hybridizing sequence substantiallycomplementary to a consensus sequence or a degenerate sequence encodingamino acid sequence of a conserved homology segment on the first cDNAstrands to hybridize therewith, under conditions in which the secondprimer anneals to the consensus sequence or degenerate sequence of firstcDNA strands, whereby 3′-end cDNA segments having the consensus sequenceor degenerate sequence are generated; and

[0330] (d) performing a second stage amplification of the 3′-end cDNAsegments generated from step (c) at a second annealing temperature whichis high stringent conditions, comprising at least two cycles of primerannealing, primer extending and denaturing, using the same first andsecond primers as used in steps (a) and (c) or a primer pair eachcomprising a nucleotide sequence corresponding to each 5′-end portion ofthe first and second primers used in steps (a) and (c), respectively,under conditions in which each primer anneals to the 3′- and 5′-endsequences of the 3′-end cDNA segments, respectively, whereby the 3′-endconserved homology cDNA segments are amplified.

[0331] This specific embodiment follows in principle, the present methodfor 3′ RACE as discussed previously except for the second primer used.

[0332] In another specific embodiment of this invention, there isprovided the method using two stage amplifications, which comprises:

[0333] (a) performing steps of (a)-(e) of the method for amplifying apopulation of full-length double-stranded cDNA, whereby full-length cDNAstrands are generated;

[0334] (b) performing a first-stage amplification of the full-lengthfirst cDNA strands obtained from step (a) at a first annealingtemperature, which comprises the steps of:

[0335] (i) at least one cycle of primer annealing, primer extending anddenaturing using a first primer comprising a nucleotide sequencesubstantially complementary to the 3′-end sequences of the full-lengthfirst cDNA strands under conditions in which the first primer anneals tothe full-length first cDNA strands, under conditions in which the firstprimer anneals to the 3′- ends of the full-length first cDNA strands,whereby full-length second cDNA strands are generated; and

[0336] (ii) at least one cycle of primer annealing, primer extending anddenaturing using a second primer of any one of claims 1-25 having at its3′ end portion a hybridizing sequence substantially complementary to aconsensus sequence or a degenerate sequence encoding amino acid sequenceof a conserved homology segment on the full-length second cDNA strandsto hybridize therewith, under conditions in which the second primeranneals to the consensus sequence or degenerate sequence of full-lengthsecond cDNA strands, whereby 5′-end cDNA segments having the consensussequence or degenerate sequence are generated; and

[0337] (c) performing a second stage amplification of the 5′-end cDNAsegments generated from step (b) at a second annealing temperature whichis high stringent conditions, comprising at least two cycles of primerannealing, primer extending and denaturing, using the same first andsecond primers as used in steps (b)-(i) and (b)-(ii), respectively, or aprimer pair each comprising a nucleotide sequence corresponding to each5′-end portion of the first and second primers used in steps (b)-(i) and(b)-(ii), respectively, under conditions in which each primer anneals tothe 3′- and 5′-end sequences of the 5′-end cDNA segments, respectively,whereby the 5′-end conserved homology cDNA segments are amplified.

[0338] This specific embodiment follows in principle, the present methodfor 5′ RACE as discussed previously except for the second primer used.

[0339] In further aspect of this invention, there is provided a methodfor identifying conserved homology segments in a multigene family fromgDNA, wherein the method comprises performing an amplification reactionusing primers, characterized in that at least one primer is derived fromany one of ACPs described above. Preferably, the primer having thestructure of ACP is one having at its 3 ′-end portion a hybridizingsequence substantially complementary to a consensus sequence or adegenerate sequence encoding amino acid sequence of a conserved homologysegment on the gDNA.

[0340] In a specific embodiment of this invention, there is provided themethod using two stage amplifications, which comprises:

[0341] (a) performing a first-stage amplification of the conservedhomology segments from the gDNA at a first annealing temperaturecomprising at least two cycles of primer annealing, primer extending anddenaturing, using the primer or the primer pair of any one of ACPsdescribed above, wherein each primer has at its 3′ end portion ahybridizing sequence substantially complementary to a consensus sequenceor a degenerate sequence encoding amino acid sequence of a conservedhomology segment on the gDNA to hybridize therewith, under conditions inwhich the primer or the primer pair anneals to the consensus sequence ordegenerate sequence of gDNA, whereby genomic DNA segments having theconsensus sequence or degenerate sequence are generated; and

[0342] (b) performing a second-stage amplification of the genomic DNAsegments generated from step (a) at a second annealing temperature,which is high stringent conditions, comprising at least one cycle ofprimer annealing, primer extending and denaturing, using the same primeror primer pair as used in step (a) or a primer or a primer pair eachcomprising a nucleotide sequence corresponding to each 5 -end portion ofthe primer or primer pair used in step (a), under conditions in whichthe primer or each of the primer pair anneals to the 3′- and 5′-endsequences of the genomic DNA segments generated from step (a),respectively, whereby the conserved homology genomic segments areamplified.

[0343] The present method follows in principle, the present method foramplifying a target nucleic acid sequence from a DNA as discussedpreviously except for the primer used.

[0344] Since this application using the ACP of this invention employs inprinciple the present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity. In addition, where an mRNA is used as starting material,the present methods for 3′ or 5′ RACE are in principle applied to thepresent methods for the identification of conserved homology segments inmultigene families.

[0345] The formula of ACP for the identification of conserved homologysegments in multigene families is identical to the formula (1) in whichthe 3′-end portion of ACP has a hybridizing sequence substantiallycomplementary to a consensus sequence in a gene family or a degeneratesequence encoding amino acid sequence of a conserved homology.

[0346] There are two principle approaches to the design of degenerateprimer: (a) using peptide sequence data obtained from a purifiedprotein; and (b) using consensus protein sequence data from alignmentsof gene families. If orthologs of the gene of interest have been clonedfrom other organisms, or if the gene is a member of a gene family, itwill be possible to generate protein sequence alignments.

[0347] These may reveal appropriate regions for the design of degenerateprimers, for example, from consensus sequence of highly conservedregions. Amplifications using degenerate primers can sometimes beproblematic and may require optimization. The first parameter isannealing temperature. It is important to keep the annealing temperatureas high as possible to avoid extensive nonspecific amplification and agood rule of thumb is to use 55° C. as a starting temperature. Ingeneral, it is difficult to keep this rule because degenerate primersshould be designed based on amino acid sequences as a precondition.However, the ACP of the present invention does not have to satisfy thisrequirement because it allows a high annealing temperature such as 65°C. at the second stage of PCR amplification regardless of primer design.

[0348] According to a preferred embodiment, the second primer is a poolof primers each comprising a degenerate sequence selected from aplurality of the nucleotides coding for amino acid sequence of theconsensus sequence.

[0349] The term “conserved region” and more specifically “conservedregion of a gene in a multigene family” as used herein refers to asegment of nucleotide sequence of a gene or amino acid sequence of aprotein that is significantly similar between members of gene families.The degree of similarity can vary. In some cases the conserved regionswill be identical between family members. In some cases the nucleotidesequence may vary significantly but still encode for amino acid segmentsthat are conserved between family members. The term “consensus sequence”as used herein refers to the bases most often found at any givenposition when comparing a large number of similar nucleotide sequences.

[0350] Alternatively, the present methods for the identification ofconserved homology segments can be also combined with that for detectingdifferentially expressed mRNAs.

[0351] In still further aspect of this invention, there is provided akit for identifying a conserved homology segment in a multigene familyby use of mRNA or gDNA, which comprises the annealing control primer orannealing control primer set described above. The descriptions of thekits for the amplification of nucleic acid sequence, 3′ RACE and 5′ RACEof this invention can be applied to the present kit.

[0352] IX. Application to Identification of a Nucleotide Variation

[0353] This application using ACP system of the subject invention canalso provide an improved method for identifying a nucleotide variationin a target nucleic acid.

[0354] In another aspect of this invention, there is provided a methodfor identifying a nucleotide variation in a target nucleic acid, whereinthe method comprises performing an amplification reaction using primers,characterized in that at least one primer is derived from any one ofACPs described above. Preferably, the primer having the structure of ACPis (a) a first primer one having at its 3′-end portion a hybridizingsequence substantially complementary to a pre-selected sequence at afirst site of target nucleic acid, wherein each of the first primer andthe first site comprises an interrogation position corresponding to thenucleotide variation, and/or (b) a second primer having a hybridizingsequence substantially complementary to a pre-selected sequence at asecond site of target nucleic acid.

[0355] In a specific embodiment of this invention, there is provided themethod using two stage amplifications, which comprises:

[0356] (a) performing a first-stage amplification to produce a first DNAstrand complementary to the target nucleic acid including the nucleotidevariation at a first annealing temperature comprising at least one cycleof primer annealing, primer extending and denaturing, using a firstprimer of any one of ACPs described above having at its 3′-end portion ahybridizing sequence substantially complementary to a pre-selectedsequence at a first site of the target nucleic acid to hybridizetherewith, wherein each of the first primer and the first site comprisesan interrogation position corresponding to the nucleotide variation,whereby the first DNA strand complementary to the target nucleic acidincluding the nucleotide variation is generated when the interrogationposition is occupied by the complementary nucleotide of the first primerto its corresponding nucleotide of the first site; and

[0357] (b) performing a second-stage amplification of the first DNAstrand generated from step (a) at a second annealing temperature, whichis high stringent conditions, comprising the steps:

[0358] (i) at least one cycle of primer annealing, primer extending anddenaturing using a second primer of any one of ACPs decribed abovehaving at its 3′-end portion a hybridizing sequence substantiallycomplementary to a pre-selected sequence at a second site of the targetnucleic acid to hybridize therewith under conditions in which the secondprimer anneals to the second site of the target nucleic acid, whereby asecond DNA strand complementary to the first DNA strand including thenucleotide variation is generated; and

[0359] (ii) at least one cycle of primer annealing, primer extending anddenaturing using the same first and second primers as used in steps (a)and (b)-(i) or a primer pair each having a hybridizing sequencecomplementary or corresponding to the 3′- and 5′-ends of the second DNAstrand generated from step (b)-(i) to hybridize therewith, underconditions in which each primer anneals to the 3′- and 5′-end sequencesof the second DNA strand, respectively, whereby the second DNA strandwhich comprises the first and second sites of the target nucleic acid atits 3′-and 5′-ends is amplified so that a short target nucleotidesegment corresponding to the second DNA strand containing the nucleotidevariation is generated.

[0360] Since this application using the ACP of this invention employs inprinciple the present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity.

[0361] A schematic representation of this specific embodiment for singlenucleotide polymorphism (SNP) genotyping using novel ACP is illustratedin FIG. 7A.

[0362] The formula of ACP for the detection of a nucleotide variation,is identical to the formula (1) in which its 3′-end portion comprises ahybridizing sequence substantially complementary to a pre-selectedsequence at a site of the target nucleic acid to hybridize therewithwhich contains the nucleotide variation, wherein the nucleotidecorresponding to the nucleotide variation and its complementarynucleotide of the ACP occupy an interrogation position. The process forthis application is carried out by two stage PCR amplifications usingthe genomic DNA obtained from samples such as patient blood or a shortsegment of the sample DNA, which includes a target nucleotide variation.The interesting nucleotide sample may be obtained from human nucleicacid and an organism that can cause an infectious disease.

[0363] The method using two-stage PCR amplifications for detectingsingle nucleotide polymorphism (SNP) genotyping basically follows theprocess used for amplifying a target nucleic acid sequence using genomicDNA as a starting material. In addition, the process for multiplex DNAamplification can be adapted to this application. To use a short segmentof the sample DNA including a target nucleotide variation as a startingmaterial for the above process, it is preferable that the target shortsegment is pre-amplified prior to step (a) using a primer pair in whicheach has a hybridizing sequence substantially complementary to thesample DNA to hybridize therewith. Furthermore, more than one targetnucleotide segment each including a SNP can be prepared by the multiplexDNA amplification as described in Application II to be used as astarting material in the subject invention for multiple SNP screening.

[0364] The first ACP used in step (a) for the detection of a polymorphicbase is an allele-specific ACP which contains an interrogation positionwithin its 3′-end portion occupied by a complementary nucleotide to thecorresponding nucleotide of the nucleotide variation in a target nucleicacid. Preferably, the interrogation position of the first primer is inthe middle of its 3′-end portion. In a more preferred embodiment, theinterrogation position of the allele-specific ACP is within about 10bases of the 3′-end nucleotide. More advantageously, the interrogationposition of the allele-specific ACP is within about 6 bases of the3′-end nucleotide of the allele-specific ACP. In another preferredembodiment, the interrogation position of the allele-specific ACP islocated within positions 4 and 6 from the 3′-end nucleotide. Mostpreferably, the interrogation position of the allele-specific ACP islocated in position 5 from the 3′-end nucleotide. The term “3 -endnucleotide” used herein refers to a nucleotide which is positioned atthe 3′-end of ACP.

[0365] In another embodiment, the 3′-end portion of the allele-specificACP used in step (a) contains at least 6 nucleotides in length, which isa minimal requirement of length for primer annealing. Preferably, the 3-end portion sequence is about 8 to 20 nucleotides in length. Mostpreferably, the 3′-end portion sequence is about 10 nucleotides inlength including an interrogation position.

[0366] In one embodiment, at least one artificial mismatch can be alsoplaced within the 3′-end portion of ACP using universal base ornon-discriminatory analog that hydrogen-bonds minimally with all fourbases without steric disruption of the DNA duplex. Although the positionof the artificial mismatch can vary depending on experimental designs,it is preferred that the mismatch nucleotide is substantially adjacentthe interrogation position of the first primer.

[0367] In a preferred embodiment, the first or second primers compriseat least one nucleotide with a label for detection or isolation.

[0368] According to a preferred embodiment, the first DNA strandincluding nucleotide variation in step (a) is generated by one cycle ofprimer annealing, primer extending, and denaturing. It is preferred thatthe second DNA strand including nucleotide variation in step (b)-(i) isgenerated by one cycle of primer annealing, primer extending, anddenaturing. Preferably, the second DNA strand including nucleotidevariation in step (b)-(ii) is amplified by at least 5 cycles of primerannealing, primer extending, and denaturing.

[0369] In another specific embodiment of this invention using amplifiedshort DNA strand fragment containing the nucleotide variation, there isprovided the method using two individual amplifications of a first and asecond amplifications in which the second amplification is performedusing two stage amplifications, which comprises:

[0370] (a) performing the first amplification to produce a short DNAstrand fragment containing the nucleotide variation between its endscomprising at least two cycles of primer annealing, primer extending anddenaturing, using a primer pair each primer comprising a hybridizingsequence substantially complementary to a pre-selected sequence at asite of the target nucleic acid under conditions that the nucleotidevariation is positioned between the pre-selected sequences, in which atleast one primer of the primer set is any one of ACPs described abovehaving at its 3′-end portion the hybridizing sequence, whereby the shortDNA strand fragment containing the nucleotide variation between its endsis amplified;

[0371] (b) performing a first-stage amplification of the secondamplification to produce a first DNA strand complementary to the shortDNA strand fragment including the nucleotide variation at a firstannealing temperature comprising at least one cycle of primer annealing,primer extending and denaturing, using a first primer of any one of ACPsdescribed above having at its 3′-end portion a hybridizing sequencesubstantially complementary to a pre-selected sequence at a first siteof the target nucleic acid to hybridize therewith, wherein each of thefirst primer and the first site comprises an interrogation positioncorresponding to the nucleotide variation, whereby the first DNA strandcomplementary to the target nucleic acid including the nucleotidevariation is generated when the interrogation position is occupied bythe complementary nucleotide of the first primer to its correspondingnucleotide of the first site; and

[0372] (c) performing a second-stage amplification of the secondamplification of the first DNA strand generated from step (a) at asecond annealing temperature, which is high stringent conditions,comprising at least one cycle of primer annealing, primer extending anddenaturing using a primer pair in which amongst the primer pair one isthe same as the primer of any one of ACPs used in step (a) the other isthe same as the first primer used in step (b), or a primer pair eachhaving a hybridizing sequence complementary or corresponding to the 3′-and 5′-ends of the first DNA strand generated from step (b) to hybridizetherewith, under conditions in which each primer anneals to the 3′- and5′-end sequences of the first DNA strand, respectively, whereby thefirst DNA strand is amplified so that a short target nucleotide segmentcorresponding to the first DNA strand containing the nucleotidevariation is generated.

[0373] A schematic representation of another specific embodiment forsingle nucleotide polymorphism (SNP) genotyping using novel ACP isillustrated in FIG. 7B. Since this specific embodiment is carried out ina similar manner to above embodiment, the common descriptions betweenthem are omitted in order to avoid the complexity of this specificationleading to undue multiplicity.

[0374] The present method can be applied to a variety of nucleotidevariations including single nucleotide polymorphism and point mutation(substitution, deletion and insertion).

[0375] The amplified products can be analyzed by gel electrophoresis. Inone embodiment, the resulting PCR products can be also detected on adenaturing polyacrylamide gel by autoradiography or non-radioactivedetection methods such as silver staining (Gottschlich et al., 1997;Kociok et al., 1998), the use of fluoresenscent-labelledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), and the use of biotinylated primers (Korn etal., 1992; Tagle et al., 1993; Rosok et al., 1996).

[0376] The amplified products generated by multiplex DNA amplificationfor multiple SNP screening can be compared through the size separationof the products. The size separation comparison is also performed byelectrophoresis through an agarose gel matrix or polyacrylamide gelmatrix or sequencing. The products can be also detected by the use offluoresenscent-labelled oligonucleotide primers for automatic analysis.

[0377] The term “interrogation position” as used herein refers to thelocation of a specific nucleotide base of interest within a targetnucleic acid. For example, in the analysis of SNPs, the “interrogationposition” in the target nucleic acid is in position what would bedifferent from wild type. The interrogation position also includes thelocation of nucleotide sequence of a primer which is complementary to aninterrogation position of the target nucleic acid. The interrogationposition of the target nucleic acid is opposite the interrogationposition of the primer, when the primer is hybridized with the targetnucleic acid.

[0378] The term “polymorphism” as used herein refers to the presence oftwo or more alternative genomic sequences or alleles between or amongdifferent genomes or individuals. “Polymorphic” refers to the conditionin which two or more variants of a specific genomic sequence can befound in a population. A “polymorphic site” is the locus at which thevariation occurs. A single nucleotide polymorphism, or SNP, is a singlebase-pair variant, typically the substitution of one nucleotide byanother nucleotide at the polymorphic site. Deletion of a singlenucleotide or insertion of a single nucleotide, also give rise to singlenucleotide polymorphisms. Typically, between different genomes orbetween different individuals, the polymorphic site may be occupied bytwo different nucleotides. The term “allele” as used herein refers aspecific member of a collection of naturally occurring sequence variants(detectable within a population of individuals) at a specific genomiclocus or marker.

[0379] In still another aspect of this invention, there is provided akit for identifying a nucleotide variation in a target nucleic acid,which comprises the annealing control primer or annealing control primerset (including the first and second primers) described above. Thedescriptions of the kits for the amplification of nucleic acid sequenceof this invention can be applied to the present kit.

[0380] X. Application to Mutagenesis

[0381] This application using ACP of the subject invention can alsoprovide an improved method for mutagenesis. The ACP-based PCR providesan excellent tool for mutagenesis, including deletion, or insertion ofsequences, the alteration of one or a few specific nucleotides, and therandom mutation of nucleotide sequence.

[0382] In further aspect of this invention, there is provided a methodfor mutagenesis in a target nucleic acid, comprising performing anamplification reaction using primers, characterized in that at least oneprimer is derived from any one of ACPs described above. Preferably, theprimer having the structure of ACP is one having at its 3′-end portion ahybridizing sequence substantially complementary to a region of targetnucleic acid sequence, wherein the hybridizing sequence has a nucleotidesequence responsible for mutagenesis.

[0383] In a specific embodiment of this invention, there is provided themethod using two stage amplifications, which comprises:

[0384] (a) performing a first-stage amplification of the target nucleicacid sequence at a first annealing temperature comprising at least twocycles of primer annealing, primer extending and denaturing, using aprimer pair of any one of ACPs described above each having at its 3′endportion a hybridizing sequence substantially complementary to a regionof the target nucleic acid sequence to hybridize therewith, wherein thehybridizing sequence has at least one mismatch nucleotide to generatesite-directed mutation, under conditions in which the primer or primerpair anneals to its target nucleotide sequence, whereby an amplificationproduct containing site-directed mutation site is generated; and

[0385] (b) performing a second-stage amplification of the amplificationproduct generated from step (a) at a second annealing temperature, whichis high stringent conditions, comprising at least one cycle of primerannealing, primer extending and denaturing, using the same primers asused in step (a) or a primer pair each comprising a pre-selectedarbitrary nucleotide sequence corresponding to each 5′-end portion ofthe primers used in step (a), under conditions in which each primeranneals to the 3′- and 5′-ends of the amplification product,respectively, whereby the amplification product containing site-directedmutation site is re-amplified.

[0386] This specific embodiment relates to site-directed mutagenesis.

[0387] Since this application using the ACP of this invention employs inprinciple the present methods for amplification of nucleic acid sequencepreviously discussed, the common descriptions between them are omittedin order to avoid the complexity of this specification leading to unduemultiplicity.

[0388] The formula of ACP for PCR mutagenesis is identical to theformula (1) in which the 3′-end portion comprises a sequence forsite-directed mutagenesis or for random mutation.

[0389] In still further aspect of this invention, there is provided akit for mutagenesis in a target nucleic acid, which comprises theannealing control primer or annealing control primer set describedabove. The descriptions of the kits for the amplification of nucleicacid sequence of this invention can be applied to the present kit.

[0390] XI. Other Applications

[0391] The ACP of the subject invention can be also useful in a varietyof processes involving nucleic acid amplifications, particularly, PCR.For example, the processes include mixed oligonucleotide-primedamplification of cDNA, long-range PCR, linear PCR, inverse PCR,quantitative PCR, touchdown PCR, sequencing, in situ PCR, vectorette PCRand thermal asymmetric interlaced PCR. The general procedures for thesemethods can be found in Joseph Sambrook, et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.(2001) and M. J. McPherson, et al., PCR, Springer-Verlag NewYork Inc., N.Y.(2000).

[0392] Therfore, the present invention encompasses all uses of theprimer according to ACP for process involving nucleic acidamplification, particularly PCR.

[0393] The ACP of this invention is significantly effective and widelyaccessible to nucleic acid amplification-based applications. Also,various problems related to primer annealing specificity remained in theprevious nucleic acid amplification techniques can be fundamentallysolved by the ACP and the methods of the present invention. The mainbenefits to be obtained from the use of the ACP during nucleic acidamplification are as follows:

[0394] (a) since the presence of a regulator portion positioned betweenthe 3′- and 5′-end portions restricts primer annealing portion to the3′-end portion under such conditions that the 3′-end portion anneals tothe template, the annealing sequence of a primer can be preciselycontrolled, which makes it possible to design a primer caplable ofhaving only a desired number of sequence annealed (or possible to designa primer capable of controlling an annealing portion thereof). It isparticularly useful when an annealing portion of a primer has to belimited (e.g., SNP genotyping, DNA microarrary screening, and detectionof differentially expressed genes);

[0395] (b) since the presence of a regulator portion positioned betweenthe 3′- and 5′-end portions interrupts the annealing of the 5′-endportion to the template under such conditions that the 3′-end portionanneals to the template, eventually the 5′-end portion not involved inthe annealing provides the 3′-end portion with primer annealingspecificity;

[0396] (c) the specificity of primer annealing is highly sensitiveenough to detect even a single-base mismatching. It is particularlyuseful for single nucleotide polymorphisms(SNPs) genotyping;

[0397] (d) the ACP is capable of providing a primer with a hightolerance in “primer search parameters” for primer design such as primerlength, annealing temperature, GC content, and PCR product length;

[0398] (e) the ACP provides two-stage nucleic acid amplifications whichallow the amplified products to be excluded from non-specificamplification;

[0399] (f) the efficiency of nucleic acid amplification is increased,which makes it easier to detect rare mRNAs; and

[0400] (g) the reproducibility of nucleic acid amplification products isincreased, which saves a great amount of time and cost.

[0401] As much as the nucleic acid amplification technology such as PCRhas influenced the biotechnological field, the use of ACP fundamentallyalter the principles of the current existing nucleic acid amplificationmethods, as mentioned above, by exhibiting unlimited applicability, andhave them significantly upgraded at one time. In consequence, the ACPand its various applications described herein provide a turning point toopen a new biotechnological era since the introduction of nucleic acidamplification technology.

[0402] The following specific examples are intended to be illustrativeof the invention and should not be construed as limiting the scope ofthe invention as defined by appended claims.

EXAMPLES

[0403] In the experimental disclosure which follows, the followingabbreviations apply to: M (molar), mM (millimolar), μM (micromolar), g(gram), μg (micrograms), ng (nanograms), l (liters), ml (milliliters),μl (microliters), ° C. (degree Centigrade); Promega (Promega Co.,Madison, U.S.A.); Clontech (CLONTECH Laboratories, Palo Alto, U.S.A.);Roche (Roche Diagnostics, Mannheim, Germany); QIAGEN (QIAGEN GmbH,Hilden, Germany). The primers used in the subject invention are shown inTable 1.

EXAMPLE 1 Evaluation of Universal Base Effect in ACP

[0404] The effect of universal base residues positioned between the 3′-and 5 -end portions of ACP was evaluated by RT-PCR using mouse conceptustissues.

[0405] Total RNA was isolated from the entire conceptuses of mousestrain ICR at the day of 4.5, 11.5 and 18.5 during gestation periodusing either Tri-reagent (Sigma), or the LiCl/Urea method (Hogan et al.,1994) as previously described (Chun et al., 1999; Hwang et al., 2000).Two individual experiments of cDNA amplifications using ACP wereperformed to examine the effect of universal base, particularly,deoxyinosine residues positioned between the 3′- and 5′-end portions ofACP as follows: A. The effect of deoxyinosine residues positionedbetween the 3 ′- and 5′-end portions of ACP in comparison with ACP andthe conventional primer not containing a dexoyinosine group; B. Theeffect of deoxyinosine residues positioned between the 3′- and 5′-endportions of ACP in association with the alteration of number ofdexoyinosine.

[0406] These experiments were conducted based on the followingassumptions:

[0407] (i) the presence of universal base residues which have lowerT_(m) than other portions in ACP due to their weaker hydrogen bondinginteractions in base pairing would not be involved in annealing to thetemplate nucleic acid under the conditions that the 3′-end portion ofACP anneals to a site of the template at a first annealing temperature.

[0408] (ii) the presence of at least one universal base residue betweenthe 3′- and 5′-end portions of ACP would be capable of interrupting theannealing of the 5′-end portion and restricting a primer annealingportion to the 3′-end.

[0409] (iii) the 3′-end portion of ACP would act only as a annealingportion to the template during PCR.

[0410] (iv) the 3′-end portion of dT-ACP which is dT₁₀ comprising 10 Tnucleotides also has too low T_(m) to bind the template nucleic acid.

[0411] (v) consequently, the dT₁₀-ACP does not produce any PCR productsunder high annealing temperature.

[0412] A. The effect of deoxyinosine residues positioned between the 3′-and 5′-end portions of ACP in comparison with ACP with the primer notcontaining a dexoyinosine group

[0413] (a) First-strand cDNA synthesis

[0414] dT₁₀-JYC2 5′-GCTTGACTACGATACTGTGCGATTTTTTTTTT-3′ (SEQ ID NO:29)or dT₁₀-ACP1 5′-GCTTGACTACGATACTGTGCGAIIIIITTTTTTTTTT-3′ (SEQ ID NO:30)was used as a cDNA synthesis primer.

[0415] Three micrograms of total RNA and 2 μl of 10 μM dT₁₀-JYC2 or 10μM dT₁₀-ACP1 were combined in a 20 μl final volume. The solution washeated at 65° C. for 10 minutes, quenched on ice, and microcentrifugedto collect solvent at the bottom. The following components were addedsequentially to the annealed primer/template on ice: 0.5 μl (40units/μl) of RNasin ribonuclease inhibitor (Promega), 4 μl of 5×reaction buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl₂, 50 mMDTT; Promega), 5 μl of 2 mM each deoxynucleotide mix (dATP, dCTP, dGTP,dTTP), and 1 μl of Moloney-murine leukemia virus (M-MLV) reversetranscriptase (200 units/μl; Promega). The 20 μl of reaction mixture wasincubated at 37° C. for 90 min, microcentrifuged, and placed on ice for2 min. The reaction was stopped by incubation at 94° C. for 2 min.

[0416] (b) cDNA amplification using ACPs

[0417] The dT₁₀-ACP1 was used to examine the effect of a deoxyinosinegroup positioned between the 3′- and 5′-end portions during PCR. ThedT₁₀-JYC2 not containing a deoxyinosine group was used as a control.

[0418] The ACP10 5′- GTCTACCAGGCATTCGCTTCATIIIIIGCCATCGACC-3′ (SEQ IDNO: 13) was used as 5′ primer for this experiment.

[0419] The PCR amplification was conducted in a 50 μl volume containing50 ng of the first-strand cDNA, 5 μl of 10× PCR buffer, 1 μl of 10 μM5′primer (ACP10), 1 μl of 10 μM 3′primer (dT₁₀-JYC2 or dT₁₀-ACP1), 3 μlof 25 mM MgCl₂, 5 μl of 2 mM dNTP, 0.5 μl of Taq polymerase (5units/μl). The PCR reactions were conducted under the followingconditions: 5 min at 94° C. followed by 30 cycles of 94° C. for 1 min,54° C. for 1 min, and 72° C. for 1 min; followed by a 5 min finalextension at 72° C. Amplified products were analyzed by electrophoresisin a 2% agarose gel followed by ethidium bromide staining.

[0420] As a result, FIG. 8 shows that the dT₁₀-ACP1 containing adeoxyinosine group produced almost no products (lanes 4-6), whereas thedT₁₀-JYC2 not containing a deoxyinosine group produced a plurality ofamplified cDNA products (lanes 1-3). Consistent with our assumption, theresults clearly indicate that the deoxyinosine group positioned betweenthe 3′-and 5′-end portions affects the annealing of the 3′- and 5′-endportions of the dT₁₀-ACP to the template cDNA under such high annealingtemperature, resulting in no product as stated in the above assumption.

[0421] B. The effect of deoxyinosine residues positioned between the 3′-and 5′-end portions of ACP in association with the alteration of numberof dexoyinosine

[0422] (a) First-strand cDNA synthesis

[0423] The first-strand cDNA was synthesized from total RNA of mouseconcentues using dT₁₀-JYC2 as a cDNA synthesis primer as the above.

[0424] (b) cDNA amplification using ACPs

[0425] This experiment used four ACPs each comprising different numberof deoxyinosine residues as follows, to examine the effect ofdeoxyinosine residues positioned between the 3′- and 5′-end portions inassociation with the alteration of number of deoxyinosine, under aparticular stringency conditions.

[0426] ACP16 5′-GTCTACCAGGCATTCGCTTCATIIGCCATCGACC-3′ (SEQ ID NO:20);

[0427] ACP17 5′-GTCTACCAGGCATTCGCTTCATIIIIGCCATCGACC-3′ (SEQ ID NO:21);

[0428] ACP18 5′-GTCTACCAGGCATTCGCTTCATIIIIIIGCCATCGACC-3′ (SEQ IDNO:22);

[0429] ACP19 5′-GTCTACCAGGCATTCGCTTCATIIIIIIIIGCCATCGACC-3′ (SEQ IDNO:23); and

[0430] CRP2I0 5′-GTCTACCAGGCATTCGCTTCATGCCATCGACC-3′ (SEQ ID NO:19) notcontaining a deoxyinosine group was used as a control.

[0431] The resultant first-strand cDNA generated from step (A), whichcomprises the pre-selected arbitrary sequence of the dT₁₀-ACP at its5′-end, was used as a template and the primer JYC25′-GCTTGACTACGATACTGTGCGA-3′ (SEQ ID NO:10) corresponding to the 5′-endportion of the dT₁₀-ACP was used as 3′ primer.

[0432] The PCR amplification was conducted in a 50 μl volume containing50 ng of the first-strand cDNA, 5 μl of 10× PCR buffer, 1 μl of 10 μM5′primer (ACP16, 17, 18, 19, or CRP2I0), 1 μl of 10 μM 3′primer (JYC2),3 μl of 25 mM MgCl₂, 5 μl of 2 mM dNTP, 0.5 μl of Taq polymerase (5units/μl). The PCR reactions were comprised of: 5 min at 94° C.,followed by 30 cycles of 94° C. for 1 min, 57° C. for 1 min, and 72° C.for 1 min; followed by a 5 min final extension at 72° C. Amplifiedproducts were analyzed by electrophoresis in a 2% agarose gel followedby ethidium bromide staining.

[0433] As a result, FIG. 9 shows that the CRP2I0 not containing anydeoxyinosine residues produced a plurality of amplified cDNA products,whereas the ACPs containing at least two deoxyinosine residues generatedthe significant reduction of amplified cDNA products, and even more, theACP containing eight deoxyinosine residues produced almost no products.Consistent with our assumption, the results clearly indicates that theannealing of the 3′-end portion of ACP to the template could beseparated from the 5′-portion since a group of contiguous deoxyinosineresidues separates the annealing of the 3′-end and 5′-end portions underhigh stringent conditions due to the property of deoxyinosine such asits weaker hydrogen bonding interaction in base pairing.

EXAMPLE 2 Method for Amplifying a Target Nucleic Acid Sequence Using ACP

[0434] The ACP of the subject invention was applied to amplify targetnucleotide sequences of mouse placenta-specific homeobox gene Esxl cDNA.The process and results for the amplification of the target nucleotidesequences of Esxl cDNA using ACPs are described herein. Total RNA (3 μg)obtained from mouse 18.5-day-old placenta was used as a startingmaterial. First-strand cDNAs were prepared under the same conditions asused in the cDNA synthesis of Example 1, except that Oligo-dT₁₅ was usedas the first-strand cDNA synthesis primer.

[0435] Oligo-dT₁₅ 5′-TTTTTTTTTTTTTTT-3′ (SEQ ID NO:54)

[0436] The resultant first-strand cDNAs were used as templates toamplify target cDNA fragments of Esxl using ACPs. These experimentsconducted two stage PCR amplifications, which is one of unique featuresof the present invention.

[0437] The conventional primers of Esxl used in the Example are: EsxN75′-GCCGGTTGCAGAAGCACC-3′; (SEQ ID NO:44) EsxC65′-GAACCATGTTTCTGAATGCC-3′; (SEQ ID NO:45) EsxN15′-GAATCTGAAACAACTTTCTA-3′; (SEQ ID NO:48) EsxC25′-GATGCATGGGACGAGGCACC-3′; (SEQ ID NO:49) EsxN35′-CGCCGCAACCCCTGCCCGCA-3′; and (SEQ ID NO:51) EsxC55′-GATGCATGGGACGAGGCA-3′. (SEQ ID NO:52)

[0438] Three primer sets, EsxN7 and EsxC6, EsxN1 and EsxC2, and EsxN3and EsxC5, were used in the Example because they are known as the primersets which generate high backgrounds as well as non-specific products inconventional PCR methods as known in the art.

[0439] According to single-target PCR systems, primers with similarmelting temperatures (T_(m) ) should be chosen. However, a primer set ofEsxN1 (T_(m) 50.7° C.) and EsxC2 (T_(m) 71.9° C.) shows about 20° C. ofdifferent melting temperatures between them, and a primer set of EsxN3(T_(m) 86.9° C.) and EsxC5 (T_(m) 66.2° C.) both has high meltingtemperatures. Also, a primer set of EsxN7 (T_(m) 68.2° C.) and EsxC6(T_(m) 61.2° C.), which has relatively similar melting temperature, areselected to observe the effect of ACP.

[0440] The ACP of the subject invention was applied to these threeconventional primer sets to demonstrate if the ACP system can overcomethe main problems arising from these conventional primer sets, such asbackground and non-specific products.

[0441] The following ACPs comprise the sequences of the aboveconventional primers at their 3′-end portions and were used as Esxlgene-specific primers for the first-stage PCR amplification: EsxN7-ACP5′ primer 5′-GTCTACCAGGCATTCGCTTCATIIIIIGCCGGTTGCAGAAGCACC-3′; (SEQ IDNO:46) EsxC6-ACP 3′ primer5′-GCTTGACTACGATACTGTGCGAIIIIIGAACCATGTTTCTGAATGCC-3′; (SEQ ID NO:47)EsxN1-ACP 5′ primer5′-GTCTACCAGGCATTCGCTTCATIIIIIGAATCTGAAACAACTTTCTA-3′; (SEQ ID NO:50)EsxC2-ACP 3′ primer5′-GCTTGACTACGATACTGTGCGAIIIIIGATGCATGGGACGAGGCACC-3′; (SEQ ID NO:55)EsxN3-ACP 5′ primer5′-GTCTACCAGGCATTCGCTTCATIIIIICGCCGCAACCCCTGCCCGCA-3′; and (SEQ IDNO:53) EsxC5-ACP 3′ primer5′-GCTTGACTACGATACTGTGCGAIIIIIGATGCATGGGACGAGGCA-3′. (SEQ ID NO:56)

[0442] The 5′-end portion sequences of the ACPs were served aspre-selected arbitrary primer sequences only for the second-stage PCRamplification: JYC2 and JYC4 5′-GTCTACCAGGCATTCGCTTCAT-3′ (SEQ IDNO:12).

[0443] During the first-stage PCR amplification, the primer set ofEsxN7-ACP and EsxC6-ACP was used as 5′ and 3′ primers, respectively, togenerate the 520-bp fragment of the Esxl cDNA, the primer set ofEsxN1-ACP and EsxC2-ACP was used as 5′ and 3′ primers, respectively, togenerate the 784-bp fragment of the Esxl cDNA, and the primer set ofEsxN3-ACP and EsxC5-ACP was used as 5′ and 3′ primers, respectively, togenerate the 483-bp fragment of the Esxl cDNA.

[0444] During the second-stage PCR amplification, JYC4 and JYC2 wereused as pre-selected arbitrary 5′ and 3′ primers, respectively (PROTOCOLA). As an alternative, the complete sequences of the ACPs, instead ofthe pre-selected arbitrary primers such as JYC4 and JYC2, can be used as5′ and 3′ primers for the second-stage PCR amplification at the highstringent conditions. In this case, it is not necessary to add thepre-selected arbitrary primers to the reaction mixture at the time of orafter the first-stage PCR reaction (PROTOCOL B).

[0445] PROTOCOL A: One-stop two-stage PCR Amplifications

[0446] (A) First-stage PCR Amplification

[0447] The first-stage PCR amplification was performed by hot start PCRmethod in which the procedure is to set up the complete reactionswithout the DNA polymerase and incubate the tubes in the thermal cyclerto complete the initial denaturation step at >90° C. Then, while holdingthe tubes at a temperature above 70° C., the appropriate amount of DNApolymerase can be pipetted into the reaction.

[0448] The first-stage PCR amplification was conducted by two cycles ofPCR consisting of annealing, extending and denaturing reaction; thereaction mixture in a final volume of 49.5 μl containing 50 ng of thefirst-strand cDNA, 5 μl of 10× PCR reaction buffer (Promega), 5 μl of 25mM MgCl₂, 5 μl of dNTP (2 mM each dATP, dCTP, dGTP, dTTP), 1.35 μl of 5′ACP (1 μM) and 1.35 μl of 3′ ACP (1 μM) is pre-heated at 94° C., whileholding the tube containing the reaction mixture at the 94° C., 0.5 μlof Taq polymerase (5 units/μl; Promega) is added into the reactionmixture; the PCR reactions comprise two cycles of 94° C for 40 sec, 60°C. for 40 sec, and 72° C. for 40 sec; followed by denaturing theamplification product at 94° C.

[0449] (B) Second-Stage PCR Amplification

[0450] The resultant cDNA product generated by the first-stage PCRamplification using Esxl gene-specific ACPs was then amplified by thefollowing second-stage PCR amplification under higher annealingtemperature. After the completion of the first-stage PCR amplification,each 1 μl of 10 μM pre-selected arbitrary primers, JYC4 and JYC2, wasadded into the reaction mixture obtained from the first-stage PCRamplification, under denaturing temperature such as at 94° C. The secondstage-PCR reaction was as follows: 35 cycles of 94° C. for 40 sec, 68°C. for 40 sec, and 72° C. for 40 sec; followed by a 5 min finalextension at 72° C.

[0451] The amplified products were analyzed by electrophoresis in a 2%agarose gel and detected by staining with ethidium bromide. Theresulting PCR products can be also detected on a denaturingpolyacrylamide gel by autoradiography or non-radioactive detectionmethods such as silver staining (Gottschlich et al., 1997; Kociok etal., 1998), the use of fluoresenscent-labelled oligonucleotides (Baueret al. 1993; Ito et al. 1994; Luehrsen et al., 1997; Smith et al.,1997), and the use of biotinylated primers (Korn et al., 1992; Tagle etal., 1993; Rosok et al., 1996).

[0452] As shown in FIGS. 10A-C, the one-stop two-stage PCRamplifications for Esxl using each primer set of EsxN7-ACP andEsxC6-ACP, EsxN1-ACP and EsxC2-ACP, and EsxN3-ACP and EsxC5-ACPgenerated a single band which corresponds to the expected size, 520-bp(FIG. 10A, lane 2), 784-bp (FIG. 10B, lane 4), and 483-bp (FIG. 10C,lane 3) of Esxl cDNA fragments, respectively. Subsequent cloning andsequence analysis of the clones confirm that the band is Esxl cDNAfragments. In contrast, the conventional primer sets, which contain thesequences corresponding only to the 3′-end portions of each ACP sets,produced non-specific products as well as high backgrounds such as DNAsmear (FIG. 10A, lane 1; FIG. 10B, lane 3; FIG. 10C, lanes 1 and 2).Since the PCR products using a ACP set comprise the pre-selectedarbitrary sequences at their 5′- and 3′-ends, additional 54-bp sequencescorresponding to the pre-selected arbitrary sequences and deoxyinosineresidues were found.

[0453]FIG. 10A shows the amplified cDNA products generated by thefollowing sets of primers; a set of EsxN7 and EsxC6 (lane 1), and a setof EsxN7-ACP and EsxC6-ACP (lane 2). PCR reactions using theconventional primer set EsxN7 and EsxC6 were as follows: 5 min at 94° C.followed by 30 cycles of 94° C. for 40 sec, 60° C. for 40 sec, and 72°C. for 40 sec; followed by a 5 min final extension at 72° C.

[0454]FIG. 10B shows the amplified cDNA products generated by a singleprimer or a primer pair as follows: the primers, EsxN1 and EsxC2, wereused in lanes 1 and 2, respectively; a combination of EsxN1-ACP andconventional primer EsxC2 was used in lanes 3; two ACPs EsxN1-ACP andEsxC2-ACP were used in lane 4. When a conventional primer set, EsxN1 andEsxC2, was used under high annealing temperature of 60° C., nospecific-target product was produced. When a primer set comprising oneACP EsxN1-ACP and a conventional primer of EsxC2 was used, atarget-specific product as well as non-specific products were amplifieddue to the non-specific binding of the conventional primer EsxC2 (lane3). However, when a ACP set was used, only a single target-specificproduct was amplified (lane 4), which indicates that the ACP of thesubject invention provides primers with tolerance to “primer designparameter” related to melting temperatures of general primers requestedfor single-target PCR systems.

[0455]FIG. 10C shows the amplified cDNA products generated by using thefollowing primer sets: a set of EsxN3 and EsxC5 was used in lanes 1 and2, and a set of EsxN3-ACP and EsxC5-ACP was used in lane 3. PCRreactions using the conventional primer set of EsxN3 and EsxC5 were asfollows: 5 min at 94° C. followed by 30 cycles of 94° C. for 40 sec, 58°C. for 40 sec, and 72° C. for 40 sec; followed by a 5 min finalextension at 72° C. (lane 1). The conventional primer set was alsocompared with the ACP set by conducting the same two stage PCRamplifications as used in the ACP, such that its annealing temperatureis increased from 60° C. to 68° C. (lane 2). These results also indicatethat although the conventional primers including ones having high T_(m)are used in the same two stage PCR amplification, they could not be freefrom the problems of non-specific products and background, whereas theACP of the subject invention can help overcome such problems arisingfrom these conventional primers.

[0456] PROTOCOL B: Non-stop two-stage PCR Amplifications

[0457] Alternatively, the complete sequences of the ACPs, instead of thepre-selected arbitrary primers such as JYC4 and JYC2, can be used asprimers for the second-stage PCR amplification at the high stringentconditions. In this case, it is not necessary to add the pre-selectedarbitrary primers to the reaction mixture at the time of or after thefirst-stage PCR reaction.

[0458] The process of the non-stop two-stage PCR amplifications isbasically identical to Protocol A, except that the ACPs, 1 μl of 5′ ACP(10 μM) and 1 μl of 3′ ACP (10 μM), are added at the first stage PCRamplification and the second stage PCR amplification immediately followsthe first stage PCR amplification without any delay because there is nostep of adding pre-selected arbitrary primers.

[0459] The amplified products were analyzed by electrophoresis in a 2%agarose gel and detected by staining with ethidium bromide. Theresulting PCR products can be also detected on a denaturingpolyacrylamide gel by autoradiography or non-radioactive detectionmethods such as silver staining (Gottschlich et al., 1997; Kociok etal., 1998), the use of fluoresenscent-labelled oligonucleotides (Baueret al. 1993; Ito et al. 1994; Luehrsen et al., 1997; Smith et al.,1997), and the use of biotinylated primers (Korn et al., 1992; Tagle etal., 1993; Rosok et al., 1996).

[0460]FIG. 10D shows the amplified cDNA products generated by thenon-stop two-stage PCR Amplifications using the following single primeror a primer pair; the primers EsxN1 and EsxC2 were used in lane 1 and 2,respectively; a pair of EsxN1 and EsxC2 was used in lane 3; and a pairof EsxN1-ACP and EsxC2-ACP was used in lane 4. When a conventionalprimer set, EsxN1 and EsxC2, was used, no specific-target product wasproduced. However, when a ACP set was used in non-stop two-stage PCRamplifications, only a single target-specific product was amplified(lane 4), which is consistent with the results of one-stop two-stage PCRAmplifications (FIG. 10B).

[0461] These examples illustrate that the ACP permits the products to befree from the background problems as well as non-specificity arisingfrom the conventional primers used in PCR methods as described in theart. It could be also understood that the ACP allows the generation ofthe specific products regardless of the design of gene-specific primers.

EXAMPLE 3 Identification and Characterization of DifferentiallyExpressed mRNAs during Mouse Embryonic Development Using ACP

[0462] The ACP of the subject invention has been applied to detectdifferentially expressed mRNAs in embryonic developments. Specifically,three different procedures and results using different stages ofconceptus total RNAs as starting materials are described herein. Theprimers used in the subject invention are shown in Table 1.

[0463] A1. PROCEDURE 1

[0464] Step (1): First-strand cDNA synthesis

[0465] The first-strand cDNAs were prepared under the same conditions asused in the cDNA synthesis of Example 1 using the dT₁₀-ACP1 orJYC-T₁₅-ACP as a cDNA synthesis primer. The resultant cDNAs werepurified by a spin colunm (PCR purification Kit, QIAGEN) to removeprimers, dNTP, and the above reagents. It is necessary to perform thepurification step prior to the determination of the cDNAs concentrationusing the UV spectroscopy at an absorbance of 260 nm. The same amount ofcDNAs from each sample was used for comparing their amplificationpatterns using the ACP system described herein.

[0466] Step (2): First-stage PCR amplification using ACP

[0467] The following ACPs were used as arbitrary ACPs (AR-ACPs) for thefirst PCR amplification: ACP3 (SEQ ID NO:3)5′-GTCTACCAGGCATTCGCTTCATIIIIIGCCATCGACS-3′; ACP5 (SEQ ID NO:5)5′-GTCTACCAGGCATTCGCTTCATIIIIIAGGCGATGCS-3′; ACP8 (SEQ ID NO:8)5′-GTCTACCAGGCATTCGCTTCATIIIIICTCCGATGCS-3′; ACP10 (SEQ ID NO:13)5′-GTCTACCAGGCATTCGCTTCATIIIIIGCCATCGACC-3′; ACP13 (SEQ ID NO:16)5′-GTCTACCAGGCATTCGCTTCATIIIIIAGGCGATGCG-3′; and ACP14 (SEQ ID NO:17)5′-GTCTACCAGGCATTCGCTTCATIIIIICTCCGATGCC-3′.

[0468] The 5′-end portion sequences of the dT₁₀-ACP1 and AR-ACPs serveas pre-selected arbitrary primer sequences only for the second-PCRamplification. The pre-selected arbitrary primers are JYC2 and JYC4.

[0469] The first-strand cDNAs produced from step (1) were amplified bythe following first-stage PCR amplification using one of AR-ACPs (ACP3,ACP5, ACP8, ACP10, ACP13, or ACP14) and the dT₁₀-ACP1 as 5′ and 3′primers, respectively. The first-stage PCR amplification was conductedin a 50 μl volume containing 50 ng of the first-strand cDNA, 5 μl of 10×PCR reaction buffer (Promega), 3 μl of 25 mM MgCl₂, 5 μl of dNTP (0.2 mMeach dATP, dCTP, dGTP, dTTP), 5 μl of 5′ primer (1 μM), 5 μl of 3′primer (1 μM), and 0.5 μl of Taq polymerase (5 units/μl; Promega). ThePCR reactions were as follows: 5 min at 94° C. followed by 20 cycles of94° C. for 1 min, 50° C. for 1 min, and 72° C. for 1 min; followed by a5 min final extension at 72° C.

[0470] The cycle of the first-stage PCR amplification can be varieddepending on the types of samples. For example, the 20 cycles of thefirst PCR amplification were used for mouse conceptus samples.

[0471] Step (3): Second-stage PCR amplification using pre-selectedarbitrary primers corresponding to the 5′-end portion sequences of ACPs

[0472] The amplified cDNA products produced from step (2) arere-amplified by the following second-stage PCR amplification using twopre-selected arbitrary primers, JYC4 and JYC2, each corresponding to the5′-end portion sequences of AR-ACP and dT₁₀-ACP1, respectively. Thesecond-stage PCR amplification was conducted in a 50 μl volumecontaining 5 μl of the first amplified cDNA products (50 μl), 5 μl of10× PCR reaction buffer (Promega), 3 μl of 25 mM MgCl₂, 5 μl of 2 mMdNTP, 1 μl of 5′ primer (10 μM), 1 μl of 3′ primer (10 μM), and 0.5 μlof Taq polymerase (5 units/μl). The PCR reactions were as follows: 5 minat 94° C. followed by 30 cycles of 94° C. for 1 min, 65° C. for 1 min,and 72° C. for 1 min; followed by a 5 min final extension at 72° C.

[0473] A2. PROCEDURE 2

[0474] The alternative procedure comprises the following steps of:

[0475] (a) providing a first sample of nucleic acids representing afirst population of mRNA transcripts and a second sample of nucleicacids representing a second population of mRNA transcripts;

[0476] (b) contacting each of the first nucleic acid sample and thesecond nucleic acid sample with a first ACP, wherein the first ACP has ahybridizing sequence substantially complementary to a region of thefirst and second population of mRNA transcripts to hybridize therewith;

[0477] (c) reverse transcribing the mRNA to which the first ACPhybridizes to produce a first population of DNA strands that arecomplementary to the mRNAs in the first nucleic acid sample to which thefirst ACP hybridizes, and a second population of DNA strands that arecomplementary to the mRNA in the second nucleic acid sample to which thefirst ACP hybridizes;

[0478] (d) purifying and quantifying the complementary DNA strandsproduced as a result of the reverse transcription step (c);

[0479] (e) synthesizing a second DNA strand complementary to each of thefirst and second populations of DNA strands using a second ACP under lowstringent conditions, by at least one PCR cycle comprising denaturing,annealing and primer extension, wherein the second ACP has a hybridizingsequence substantially complementary to the first and second populationsof DNA strands;

[0480] (f) amplifying each second DNA strand obtained from step (e)under high stringent conditions, by at least one PCR cycle comprisingdenaturing, annealing and primer extension to generate first and secondpopulations of amplification products using two pre-selected arbitraryprimers each comprising a sequence corresponding to each 5′-end portionof the first and second annealing control primers; and

[0481] (g) comparing the amount of individual amplification products inthe first and second populations of amplification products.

[0482] The first-strand cDNAs are synthesized using JYC5-T₁₅-ACP 5′-CTGTGAATGCTGC GACTACGATIIIIIITTTTTTTTTTTTTTT-3′ (SEQ ID NO:61).

[0483] The 5′-end portion sequence of the JYC5-T₁₅-ACP serves as a 3′pre-selected arbitrary primer sequence to be used only for the secondstage of PCR amplification:

[0484] JYC5 5′-CTGTGAATGCTGCGACTACGAT-3′ (SEQ ID NO:60).

[0485] Step (1): First-strand cDNA synthesis

[0486] 1. Combine 3 μg total RNA and 2 μl of 10 μM JYC5-T₁₅-ACP in asterile 0.2 ml microcentrifuge tube.

[0487] 2. Add sterile H₂O to a final volume of 9.5 μl. Mix contents andspin the tube briefly in a microcentrifuge.

[0488] 3. Incubate the tube at 80° C. for 3 minutes or use athermocycler for the same purpose.

[0489] 4. Cool the tube on ice for 2 minutes. Spin down the contents ofthe tube briefly in a microcentrifuge.

[0490] 5. To the same reaction tube add the following reagents: 4 μl 5×First-strand buffer (Promega), 5 μl dNTP (2 mM each dATP, dCTP, dGTP,dTTP), 0.5 μl RNasin inhibitor (40 units/μl, Promega) and 1 μl M-MLVreverse transcriptase (200 U/μl).

[0491] 6. Mix contents and spin the tube briefly in a microcentrifuge.

[0492] 7. Incubate the tube at 42 ° C. for 90 min.

[0493] 8. Incubate the tube at 94 ° C. for 2 minutes to terminatefirst-strand synthesis.

[0494] 9. Place the tube on ice for 2 min.

[0495] 10. Purify the resultant cDNAs by a spin column (PCR purificationKit, QIAGEN) to remove primers, dNTP, and the above reagents.

[0496] 11. Next, measure the concentration of the cDNAs using the UVspectroscopy at an absorbance of 260 nm.

[0497] 12. Process to step 2.

[0498] Step (2): Second-strand cDNA synthesis using ACP

[0499] The same amount of cDNAs from each sample was used for thecomparison of their amplification patterns using the ACPs describedherein. The second-strand cDNA was synthesized using arbitrary ACP10 byhot start PCR method in which the procedure is to set up the completereactions without the DNA polymerase and incubate the tubes in thethermal cycler to complete the initial denaturation step at >90° C.Then, while holding the tubes at a temperature above 90° C., theappropriate amount of DNA polymerase can be pipetted into the reaction.

[0500] 1. Combine the following reagents in a sterile 0.2 mlmicrocentrifuge tube: 49.5 μl of the total volume containing 1 μl offirst-strand cDNA (50 ng/μl) prepared by step 1, 5 μl of 10× PCR buffer(Roche), 5 μl of 2 mM dNTP, 1 μl of 10 μM arbitrary ACP (5′ primer) and37.5 μl of sterile dH₂O.

[0501]2. Mix contents and spin the tube briefly in a microcentrifuge.

[0502] 3. Place the tube in the preheated thermal cycler at 94° C.

[0503] 2 5 4. Add the 0.5 μl of Taq polymerase (5 units/μl; Roche) intothe reaction, while holding the tube at the temperature 94° C.

[0504] 5. Conduct PCR reaction under the following conditions: one cycleof 94° C. for 5 min, 50° C. for 3 min, and 72° C. for 1 min; followed bydenaturing the first amplification product at 94° C.

[0505] Step (3): PCR Amplification of the second-strand cDNAs usingpre-selected arbitrary primers corresponding to the 5′-end portionsequences of ACPs

[0506] 1. After the completion of the first stage PCR amplification,while holding the tubes at a temperature above 94° C., add 2 μl of 10 μMJYC4 and 2 μl of 10 μM JYC5, in which each corresponds to the 5′-endportion sequences of both 5′ and 3′ ACPs, respectively, into thereaction mixture used in step (2).

[0507] 2. Conduct second stage PCR reactions under the followingconditions: 40 cycles of 94° C. for 40 sec, 68° C. for 40 sec, and 72°C. for 40 sec; followed by a 5 min final extension at 72° C.

[0508] A3. PROCEDURE 3

[0509] As an alternative process, in the step (f) of PROCEDURE 2 thecomplete sequences of the first and second ACPs used in the steps (b)and (e) of PROCEDURE 2, instead of the pre-selected arbitrary sequencesof the 5′ -end portions of the first and second ACPs, can be used as 3′and 5′ primers, respectively, at the high stringent conditions foramplifying each second DNA strand obtained from the step (e) ofPROCEDURE 2, wherein the 3′- and 5′-ends of the second DNA strands whichwere initially synthesized using the second ACP comprise the sequence ofthe first ACP and the complementary sequence of the second ACP,respectively, and also serve as perfect pairing sites to the first andsecond ACPs. In this case, it is not necessary to add the pre-selectedarbitrary primers to the reaction mixture at the time of or afterfirst-stage PCR reaction.

[0510] Step (1): First-strand cDNA synthesis

[0511] The first-strand cDNAs were prepared under the same conditions asused in the cDNA synthesis of PROCEDURE 2 using the JYC5-T₁₅-ACP as acDNA synthesis primer.

[0512] Step (2): Second-strand cDNA synthesis and amplification usingnon-stop two-stage PCR

[0513] The same amount of cDNAs from each sample was used for thecomparison of their amplification patterns using the ACPs describedherein. The second-strand cDNA was synthesized using arbitrary ACP10 byhot start PCR method in which the procedure is to set up the completereactions without the DNA polymerase and incubate the tubes in thethermal cycler to complete the initial denaturation step at >90° C.Then, while holding the tubes at a temperature above 90° C., theappropriate amount of DNA polymerase can be pipetted into the reaction.

[0514] 1. Combine the following reagents in a sterile 0.2 mlmicrocentrifuge tube: 49.5 μl of the total volume containing 1 μl offirst-strand cDNA (50 ng/μl) prepared by step 1, 5 μl of 10× PCR buffer(Roche), 5 μl of 2 mM dNTP, 1 μl of 10 μM arbitrary ACP10 (5′ primer), 1μl of 10 μM JYC5-T₁₅-ACP (3′ primer) and 36.5 μl of sterile dH₂O.

[0515] 2. Mix contents and spin the tube briefly in a microcentrifuge.

[0516] 3. Place the tube in the preheated thermal cycler at 94° C.

[0517] 4. Add the 0.5 μl of Taq polymerase (5 units/μl; Roche) into thereaction, while holding the tube at the temperature 94° C.

[0518] 5. Conduct PCR reaction under the following conditions: one cycleof 94° C. for 1 min, 50° C. for 3 min, and 72° C. for 1 min; followed by40 cycles of 94° C. for 40 sec, 65° C. for 40 sec, and 72° C. for 40sec; and followed by a 5 min final extension at 72° C.

[0519] B. Separation of amplified PCR products by electrophoresisanalysis and recovery of the differentially displayed bands

[0520] The amplified products were analyzed by electrophoresis in a 2%agarose gel and detected by staining with ethidium bromide. Severalmajor bands differentially expressed during embryonic development (E4.5,E11.5, and E18.5) were selected, excised and extracted from the gelsusing GENECLEAN II Kit (BIO 101). The resulting PCR products can be alsodetected on a denaturing polyacrylamide gel by autoradiography ornon-radioactive detection methods such as silver staining (Gottschlichet al., 1997; Kociok et al., 1998), the use of fluoresenscent-labelledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), and the use of biotinylated primers (Korn etal., 1992; Tagle et al., 1993; Rosok et al., 1996).

[0521] C. Re-amplification of the recovered bands

[0522] The bands obtained from step B were re-amplified using the samepre-selected arbitrary primers and PCR conditions as used in PROCEDURE1, 2 and 3.

[0523] D. Cloning and sequencing of the re-amplified fragments

[0524] Each amplified fragment was cloned into the pGEM-T Easy vector(Promega) and sequenced with the ABI PRISM 310 Genetic Analyzer (PerkinElmer Biosystem) using BigDye Terminator cycle sequencing kit (PerkinElmer). Computer-assisted sequence analysis was carried out using theBLAST search program (Basic Local Alignment Search Tool).

[0525] E. Northern analysis

[0526] Twenty micrograms of total RNA from conceptus tissues wereresolved on denaturing 1% agarose gels containing formaldehyde,transferred onto nylon membranes (Hybond-N, Amersham, U.S.A.), andhybridized with a ³²P-labeled subcloned PCR product in QuikHyb solution(Stratagene, U.S.A.) overnight at 58° C. as previously described (Chunet al., 1999; Hwang et al., 2000). Blots were washed at 65° C. twice for20 min in 2× SSC, 0.1% SDS, twice for 20 min in 1× SSC, 0.1% SDS, andtwice for 20 min in 0.1×SSC, 0.1% SDS. The membranes were exposed toKodak X-Omat XK-1 film with a Fuji intensifying screen at −80° C.

[0527] FIGS. 11A-D shows the amplified cDNA products, wherein mouseconceptus samples obtained from different stages were amplified byPROCEDURE 1 using the primer sets as follows; a set of ACP3 anddT₁₀-ACP1 for the lanes 1-3 of FIG. 11A; a set of ACP5 and dT₁₀-ACP1 forthe lanes 1-6 and of FIG. 11B and a set of ACP8 and dT₁₀-ACP1 for thelanes 7-12 of FIG. 10B, respectively. FIG. 11B also shows additionalresults of the amplified cDNA products generated by using another ACPsets. FIGS. 11C-D shows the amplified products generated by using twoprimer sets of the ACP10 and dT₁₀-ACP1(FIG. 11C), and ACP14 anddT₁₀-ACP1(FIG. 11D), respectively. Many differentially expressed bandsin a specific stage were obtained, subcloned into the pGEM-T Easy vector(Promega), and sequenced. Sequence analysis reveals that all of theclones are known genes except two novel genes (Table 2). The expressionpatterns were confirmed by Northern blot analysis using mouse conceptusstage blot (Seegene, Inc., Seoul, Korea).

[0528]FIG. 12A shows the amplified cDNA products, wherein mouseconceptus samples (E4.5: lane 1; E11.5: lane 2; E18.5: lane 3) obtainedfrom different stages were amplified by PROCEDURE 2 using a set of ACP10and JYC5-T₁₅-ACP. Many differentially expressed bands in a specificstage were obtained, subcloned into the pGEM-T Easy vector (Promega),and sequenced. Sequence analysis reveals that all of the clones areknown genes except one DEG 2 (Table 2). The expression patterns wereconfirmed by Northern blot analysis using mouse conceptus stage blot(Seegene, Inc., Seoul, Korea).

[0529]FIG. 12B shows the amplified cDNA products, wherein mouseconceptus samples at the different stages of (E4.5: lane 3; E11.5: lane4; E18.5: lane 5) were amplified by non-stop two-stage PCRamplifications using a set of ACP10 and JYC5-T₁₅-ACP as above mentionedin PROCEDURE 3. When a set of ACP10 and JYC5-T₁₅-ACP (lanes 3-5) wasused, the resultant bands were identical to the bands which wereobtained by PROCEDURE 2 comprising one-stop two-stage PCR amplifications(FIG. 12A). However, no products were generated when a single primer,ACP10 (lane 1) or JYC5-T₁₅-ACP (lane 2) was used, which indicates thatthe amplified products were generated only when both ACP10 andJYC5-T₁₅-ACP as a set were used for their specific bindings.

[0530]FIG. 13 shows the results of Northern blot hybridization forrepresenting six different clones using DEG1 (A; arrow 1 of FIG. 10A,FIG. 11, and FIG. 12), DEG2 (C), DEG3 (B; arrow 2 of FIG. 10A, FIG. 11,and FIG. 12), DEG5 (E), DEG7 (F), and DEG8 (D; arrow 4 of FIG. 10A, FIG.11, and FIG. 12) as probes. The DEG1 probe was also hybridized to thealternative isoform of Tropomyosin 2 (arrow 1′ of FIG. 11, and FIG. 12),which was discovered by this present invention. Consistent with theresults of agarose gel analysis, Northern blot analysis showed that theexpression patterns of the clones are identical to the original bands onthe agarose gels, indicating that all of the clones are true positiveproducts. Thus, the ACP produces only positive products without anyfalse positives, which means that the ACP eliminates the problem offalse positives.

[0531]FIG. 14 shows the results of Northern blot hybridization for theexpression of DEG5 during mouse embryonic development. DEG5, which isturned out as a novel gene by sequence analysis, shows an interestingexpression patterns: after a strong expression appeared in the earlypregnancy stage (E4.5), its expression was gradually reduced in themiddle stages and gradually increased again in the late developmentstage (E17.5 and E18.5).

[0532] These results indicate that the method using ACP for isolatingdifferentially expressed genes produces only real PCR products andcompletely eliminates false positive products.

[0533] Freedom from false positives which have been one major bottleneckremaining for the previous Differential Display technique allowsavoiding the subsequent labor-intensive work required for theverification of the cDNA fragments identified by Differential Display.

EXAMPLE 5 Method for Rapid Amplification of 3′-Ends of cDNA (3′-RACE)Using ACP

[0534] The present example compares the ACP-based 3′-RACE and theconventional 3′-RACE in order to demonstrate if the ACP of the presentinvention can exclude such background problems arising from conventionaloligo-dT primers used in cDNA synthesis.

[0535] In the conventional 3′-RACE, the poly(A) tail of mRNA moleculesis exploited as a priming site for PCR amplification and thus theoligo-dT primer is used as a 3′ primer for the conventional 3′-RACE. Incontrast, the ACP of the present invention uses the poly(A) tail of mRNAas a priming site only for the cDNA synthesis but not for the subsequentPCR amplification.

[0536] Mouse first-strand cDNAs were prepared under the same conditionsas used in the cDNA synthesis of Example 1 using Oligo VdT₁₅-ACP 5′-GCTTGACTACGATACTGTGCGAIIIII TTTTTTTTTTTTTTTV-3′ (SEQ ID NO:57) (V is A,C or G) as a cDNA synthesis primer and then, directly used as templatesfor the subsequent PCR amplification without the purification step forthe removal of the cDNA synthesis primer.

[0537] For the conventional 3′-RACE, the first-strand cDNAs weresynthesized using the following cDNA synthesis primer;

[0538] CDS III/3′ 5′-ATTCTAGAGGCCGAGGCGGCCGACATG-(dT)₃₀-VN-3′ (SEQ IDNO:35) (V is A, C or G; and N is A, C, T or G).

[0539] This cDNA synthesis primer, CDS III/3′, was used as 3′ primer forsubsequent PCR amplification.

[0540] The PCR amplification was conducted in a 50 μl volume containing50 ng of the first-strand cDNA, 5 μl of 10× PCR buffer (Promega), 1 μlof a gene-specific 5′ primer (10 μM), 1 μl of pre-selected arbitrary 3′primer JYC2 (10 μM) or CDS III/3′ (10 μM), 3 μl of 25 mM MgCl₂, 5 μl of2 mM dNTP, 0.5 μl Taq polymerase (5 units/μl; Promega). The PCRreactions were conducted under the following conditions: 5 min at 94° C.followed by 30 cycles of 94° C. for 1 min, 65° C. for 1 min, and 72° C.for 1 min; followed by a 5 min final extension at 72° C. Amplifiedproducts were analyzed by electrophoresis in a 2% agarose gel followedby ethidium bromide staining. The resulting PCR products can be alsodetected on a denaturing polyacrylamide gel by autoradiography ornon-radioactive detection methods such as silver staining (Gottschlichet al., 1997; Kociok et al., 1998), the use of fluoresenscent-labelledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), and the use of biotinylated primers (Korn etal., 1992; Tagle et al., 1993; Rosok et al., 1996).

[0541]FIG. 15 shows the results of beta-actin 3′-RACE. The conventional3′-RACE (lane 1) was compared with ACP-based 3′-RACE (lane 2). Theconventional 3′-RACE method produced non-specific products as well asDNA smear background, whereas the ACP-based 3′-RACE produced only asingle band, which is the expected size of 348-bp. These resultsindicate that the ACP-based 3′-RACE can exclude the background problemssuch as DNA smear and non-specific products.

EXAMPLE 6 Method for Rapid Amplification of 5′-End (5′-RACE) andFull-length cDNAs Using ACP

[0542] The ACP of the subject invention was also used to amplify the5′-ends of cDNA fragments. The first-strand cDNAs were synthesized usingOligo VdT₁₅-ACP, or Random dN₆-ACP:

[0543] Oligo VdT₁₅-ACP5′-GCTTGACTACGATACTGTGCGAIIIIITTTTTTTTTTTTTTTV-3′(SEQ ID NO:57), whereinV can be A, C, or G;

[0544] Random dN₆-ACP 5′-GCTTGACTACGATACTGTGCGAIIIIINNNNNN-3′ (SEQ IDNO: 58), wherein N can be A, C, G, or T.

[0545] After the complete synthesis of the first strand cDNA sequencespresent in the form of mRNA-cDNA intermediates, cytosine residues aretailed at the 3 ′-end of the first strand cDNA sequences by the terminaltransferase reaction of reverse transcriptase in the presence ofmanganese. The 3′-ends of the first strand cDNAs were extended using thefirst strand cDNA 3′-end extending ACP (rG3-ACP, rG2-ACP, or dG3-ACP)and then, directly used as templates for the subsequent PCRamplification without a purification step for the removal of the firststrand cDNA 3′-end extending ACP as well as the cDNA synthesis primer.

[0546] The sequences of the first-strand cDNA 3′-end extending ACPs are:

[0547] rG3-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIIGGr(GGG)-3′ (SEQ ID NO:36);

[0548] rG2-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIIGGr(GG)-dG-3′ (SEQ IDNO:37);

[0549] rG1-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIIGGr(G)-d(GG)-3′ (SEQ IDNO:59); or

[0550] dG3-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIIGGd(GGG)-3′ (SEQ ID NO:38)(wherein r and d represent ribonucleotide and deoxyribonucleotide,respectively).

[0551] A. First-strand full-length cDNA synthesis

[0552] PROTOCOL A: First-strand cDNA synthesis using the ACP of thesubject invention

[0553] 1. Combine the followings in a sterile 0.2 ml microcentrifugetube: 3 μg of total RNA and 2 μl of 10 μM of Oligo VdT₁₅-ACP or randomdN₆-ACP.

[0554] 2. Add sterile H₂O to a final volume of 10 μl. Mix contents andspin the tube briefly in a microcentrifuge.

[0555] 3. Incubate the tube in a 65° C. water bath for 15 minutes or usea thermocycler for the same purpose.

[0556] 4. Cool the tube on ice for at least 2 minutes. Spin down thecontents of the tube briefly in a microcentrifuge.

[0557] 5. Add the following reagents to the same reaction tube: 4 μl of5× first-strand buffer (Invitrogen), 1 μl of 0.1M DTT, 2 μl of BSA (1mg/ml), 2 μl of dNTP (10 mM each dATP, dCTP, dGTP, dTTP), 0.4 μl of 100mM MnCl₂ and 0.5 μl of RNasin inhibitor (40 units/μl, Promega).

[0558] 6. Mix contents and spin the tube briefly in a microcentrifuge.

[0559] 7. Incubate the tube at 42° C. for 2 minutes in an incubator orthermocycler.

[0560] 8. Add 1 μl of SuperScript II reverse transcriptase (200units/μl; Invitrogen).

[0561] 9. Incubate the tube at 42° C. for 1 hour in an incubator orthermocycler.

[0562] 10. Add 1 μl of 10 μM first strand cDNA 3′-end extending ACP(rG3-ACP, rG2-ACP, or dG3-ACP).

[0563] 11. Add 0.3 μl of SuperScript II reverse transcriptase (200units/μl; Invitrogen).

[0564] 12. Incubate the tube at 42° C. for 30 minutes in an incubator orthermocycler.

[0565] 13. Incubate the tube at 70° C. for 15 minutes in an incubator orthermocycler to terminate first-strand synthesis.

[0566] 14. Place the tube on ice or can be stored at −20° C.

[0567] PROTOCOL B: First-strand full-length cDNA synthesis by CapFindermethod

[0568] The following primers are used in the CapFinder method(Clontech): SMART IV ™ Oligonucleotide (SEQ ID NO:33)5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCr(GGG)-3′; and 5′ PCR primer (SEQID NO:34) 5′-AAGCAGTGGTATCAACGCAGAGT-3′, and CDS III/3′ PCR primer.

[0569] 1. Combine the followings in a sterile 0.2 ml microcentrifugetube: 3 μg of total RNA, 1 μl of 10 μM CDS III/3′ PCR primer (Clontech)and 1 μl of 10 μM SMART IV Oligonucleotide (Clontech).

[0570] 2. Add sterile H₂O to a final volume of 5 μl. Mix contents andspin the tube briefly in a microcentrifuge.

[0571] 3. Incubate the tube at 72° C. for 2 minutes.

[0572] 4. Cool the tube on ice for 2 minutes. Spin down the contents ofthe tube briefly in a microcentrifuge.

[0573] 5. Add the following reagents to the same reaction tube: 10 μl ofthe total volume containing 2 μl of 5× first-strand buffer (Clontech), 1μl of 20 mM DTT, 1 μl of dNTP (10 mM each dATP, dCTP, dGTP, dTTP) and 1μl of PowerScript Reverse Transcriptase (Clontech).

[0574] 6. Mix contents and spin the tube briefly in a microcentrifuge.

[0575] 7. Incubate the tube at 42° C. for 1 hour

[0576] 8. Place the tube on ice or can be stored at −20° C.

[0577] B. PCR amplification

[0578] PROTOCOL C: Amnplification of a target 5′-end cDNA fragment usingACP system or conventional 5′-RACE method

[0579] The present example compares the current CapFinder 5′-RACEtechnology and ACP-based 5′ RACE method, wherein the current CapFinder5′-RACE technology could not exclude the high background due to residualamount of the primers during the process. In order to demonstrate if theACP of the present invention can eliminate such background problemsarising from primers such as the CapFinder primer, SMART IVOligonucleotide (Clontech), and cDNA synthesis primer, CDS III/3′ PCRprimer (Clontech), used in cDNA synthesis, both the ACP-based 5′-RACEand the CapFinder 5′-RACE for the amplification of 5′-ends of mouse JunBand beta-actin cDNAs were conducted in the same conditions. The mouseJunB mRNA is a relatively rare transcript in mouse 18.5-day-old placentaRNA, whereas mouse beta-actin is a relatively abundant.

[0580] 1. Combine the following reagents in a sterile 0.2 mlmicrocentrifuge tube: 50 μl of the total volume containing 1 μl offirst-strand cDNA prepared from Protocol A or B, 5 μl of 10× PCR buffer(Promega), 5 μl of 25 mM MgCl₂, 5 μl of 2 mM dNTP, 1 μl of 10 μMgene-specific 5′-RACE primer, 1 μl of 10 μM JYC2 or 5′ PCR primer(Clontech), 0.5 μl of Taq Polymerase (5 units/μl; Promega) and 31.5 μlof sterile dH₂O.

[0581] 2. Mix contents and spin the tube briefly in a microcentrifuge.

[0582] 3. Conduct PCR reaction under the following conditions: 5 min at94° C., followed by 30 cycles of 94° C. for 40 seconds, 58° C. for 40seconds, and 72° C. for 1 min 30 sec; followed by a 5 min finalextension at 72° C.

[0583] 4. Analyze the amplified products by electrophoresis in a 2%agarose gel followed by ethidium bromide staining.

[0584] The resulting PCR products can be also detected on a denaturingpolyacrylamide gel by autoradiography or non-radioactive detectionmethods such as silver staining (Gottschlich et al., 1997; Kociok etal., 1998), the use of fluoresenscent-labelled oligonucleotides (Baueret al. 1993; Ito et al. 1994; Luehrsen et al., 1997; Smith et al.,1997), and the use of biotinylated primers (Korn et al., 1992; Tagle etal., 1993; Rosok et al., 1996).

[0585] As shown in FIG. 16, the CapFinder methods for mouse JunB andbeta-actin 5′ -RACE using the 5′ PCR primer (Clontech) and thegene-specific primer produced high backgrounds such as DNA smear (lanes1 and 3) as described by many researchers (Chenchik et al., 1998; Matzet al., 1999; Schramm et al., 2000), whereas the ACP-based 5′-RACE ofthe present invention generated only a single band which correspondseach to the expected size 155-bp or 319-bp of mouse JunB (lane 2) ormouse beta-actin (lane 4) 5′-end cDNA fragment, respectively. Theseexamples illustrate that the ACP can be used to fundamentally eliminatesuch background problems arising from contamination of primers usedduring cDNA synthesis, without the purification step for the removal ofprimers used in the cDNA synthesis.

[0586]FIG. 17 also shows that the ACP of the subject invention permitsthe non-specific products not to be formed, which are generated by theCapFinder method (lane 1). The first-strand cDNA was synthesized eitherby CapFinder method (lane 1) or ACP method (lanes 2, 3, and 4) and then,directly used as template in the subsequent PCR amplification for mouseprolactin-like protein PLP-C alpha 5′-RACE. The PLP-C alpha-specific5′-RACE primer is: PLP-C alpha 5′-GAGAGGATAGTTTCAGGGAC-3′ (SEQ IDNO:40). The first-strand cDNA 3′-end extending ACPs comprising eitherthree riboguanines (rG3-ACP; lane 2), three deoxyriboguanines (dG3-ACP;lane 4), or a combination of two riboguanines and one deoxyriboguanine(rG2-ACP; lane 3) at the 3′-end generated 5′-end cDNAs so that a singleband which corresponds to the expected size 506-bp of mouse PLP-C alpha5′-end cDNA fragment was produced from the ACP-based PCR for PLP-C alpha5′-RACE.

[0587] PROTOCOL D: Amplification of 5′ enriched cDNA fragments using ACP

[0588] The first-strand cDNAs are synthesized using Random dN₆-ACP inProtocol A. The PCR amplification was performed by hot start PCR methodin which the procedure is to set up the complete reactions without theDNA polymerase and incubate the tubes in the thermal cycler to completethe initial denaturation step at >90° C. Then, while holding the tubesat a temperature above 70° C., the appropriate amount of DNA polymerasecan be pipetted into the reaction.

[0589] 1.Combine the following reagents in a sterile 0.2 mlmicrocentrifuge tube: 49.5 μl of the total volume containing 1 μl offirst-strand cDNA prepared by Random dN₆-ACP in Protocol A, 5 μl of 10×PCR buffer (Promega), 5 μl of 25 mM MgCl₂, 5 μl of 2 mM dNTP, 1 μl of 10μM JYC2 (3′primer), 1 μl of 10 μM JYC4 (5′primer) and 31.5 μl steriledH₂O.

[0590] 2. Mix contents and spin the tube briefly in a microcentrifuge.

[0591] 3. Place the tube in the preheated thermal cycler at 94° C.

[0592] 4. Add the 0.5 μl of Taq polymerase (5 units/μl; Promega) intothe reaction, while holding the tube at the temperature 94° C.

[0593] 5. Conduct PCR reaction under the following conditions: 5 min at94° C. followed by 30 cycles of 94° C. for 40 seconds, 68° C. for 40seconds, and 72° C. for 1 min 30 sec; followed by a 5 min finalextension at 72° C.

[0594] 6. Analyze the amplified products by electrophoresis in a 2%agarose gel followed by ethidium bromide staining.

[0595] The resulting PCR products can be also detected on a denaturingpolyacrylamide gel by autoradiography or non-radioactive detectionmethods such as silver staining (Gottschlich et al., 1997; Kociok etal., 1998), the use of fluoresenscent-labelled oligonucleotides (Baueret al. 1993; Ito et al. 1994; Luehrsen et al., 1997; Smith et al.,1997), and the use of biotinylated primers (Korn et al., 1992; Tagle etal., 1993; Rosok et al., 1996).

[0596] PROTOCOL E: Amplification of full-length enriched cDNAs using ACP

[0597] The first-strand cDNAs are synthesized using Oligo VdT₁₅-ACP inProtocol A. The PCR amplification was performed by hot start PCR methodas in Protocol D.

[0598] 1. Combine the following reagents in a sterile 0.2 mlmicrocentrifuge tube: 49.5 μl of the total volume containing 1 μl offirst-strand cDNA prepared by Oligo VdT₁₅-ACP in Protocol A, 5 μl of 10×PCR buffer (Promega), 5 μl of 25 mM MgCl₂, 5 μl of 2 mM dNTP, 1 μl of 10μM JYC2 (3′ primer), 1 μl of 10 μM JYC4 (5′ primer) and 31.5 μl ofsterile dH₂O.

[0599] 2. Mix contents and spin the tube briefly in a microcentrifuge.

[0600] 3. Place the tube in the preheated thermal cycler at 94° C.

[0601] 4. Add the 0.5 μl of Taq polymerase (5 units/μl; Promega,Madison, U.S.A.) into the reaction, while holding the tube at thetemperature 94° C.

[0602] 5. Conduct PCR reaction under the following conditions: 5 min at94° C. followed by 30 cycles of 94° C. for 40 seconds, 68° C. for 40seconds, and 72° C. for 1 min 30 sec; followed by a min final extensionat 72° C.

[0603] 6. Analyze the amplified products by electrophoresis in a 2%agarose gel followed by ethidium bromide staining.

[0604] The resulting PCR products can be also detected on a denaturingpolyacrylamide gel by autoradiography or non-radioactive detectionmethods such as silver staining (Gottschlich et al., 1997; Kociok etal., 1998), the use of fluoresenscent-labelled oligonucleotides (Baueret al. 1993; Ito et al. 1994; Luehrsen et al., 1997; Smith et al.,1997), and the use of biotinylated primers (Korn et al., 1992; Tagle etal., 1993; Rosok et al., 1996).

[0605] To evaluate the efficiency of the method using ACP in theamplification of full-length cDNAs, the full-length cDNAs amplified byeither the above procedures of ACP method or the current CapFindermethod were blotted to a Hybond-N membrane (Amersham/United StatesBiochemical). The mouse glyceraldehydes-3-phosphate dehydrogenase(GAPDH) cDNA was labeled with [alpha-³²P]dCTP using a random labelingkit (Roche Diagnostics Co, Indianapolis, U.S.A.) and used as a probe.

[0606] As shown in FIG. 18, the GAPDH cDNA probe detected a single bandwhich corresponds to the expected size 1.3-kb of full-length GAPDI cDNA.As expected, the signals of the PCR products generated by the above ACPmethod (lane 2) were several fold stronger than the ones by theCapFinder method (lane 1). This example illustrates that the ACP methodof the present invention much more effectively amplifies full-lengthcDNAs than the CapFinder method does.

EXAMPLE 7 Genomic Fingerprinting Using ACP-based Arbitrarily Primed PCR

[0607] The ACP of the subject invention has been applied to detectpolymorphisms in mouse. The genomic DNAs of mouse strains C57BL/6J, CBA,BALB/cJ, NOR, SPRETUS, PANCEVO, and Korean Wild Mouse were used startingmaterials. Genomic DNA was prepared from the liver of mice using theQIAamp Tissue Kit (QIAGEN, Hilden, Germany).

[0608] The arbitrary ACPs used in the subject invention are: ACP101 (SEQID NO:64) 5′-GTCTACCAGGCATTCGCTTCATIIIIICCGGAGGATC-3′; ACP109 (SEQ IDNO:65) 5′-GTCTACCAGGCATTCGCTTCATIIIIICTGCAGGACG-3′; and ACP116 (SEQ IDNO:66) 5′-GTCTACCAGGCATTCGCTTCATIIIIICGGAGCATCC-3′.

[0609] A set of arbitrary ACPs, ACP101 and ACP109 (FIG. 19A), or ACP101and ACP116 (FIG. 19B), was used as primers for mouse genomicfingerprinting. The PCR amplification was performed by hot start PCRmethod as described in Example 2. The genomic fingerprinting using ACPis conducted by two stages of PCR amplifications under the followingconditions:

[0610] amplification reactions are performed under low stringentconditions by two cycles of the first-stage PCR comprising annealing,extending and denaturing reaction; the reaction mixture in the finalvolume of 49.5 μl containing 50 ng of the genomic DNA, 5 μl of 10× PCRreaction buffer (Promega), 5 μl of 25 mM MgCl₂, 5 μl of dNTP (2 mM eachdATP, dCTP, dGTP, dTTP), each 7 μl of a pair of ACPs (each 10 μM) ispre-heated at 94° C., while holding the tube containing the reactionmixture at the 94° C., 0.5 μl of Taq polymerase (5 units/μl; Promega) isadded into the reaction mixture; the PCR reactions are as follows: twocycles of 94° C. for 40 sec, 52° C. for 3 min, and 72° C. for 1 min;followed by denaturing the amplification product at 94° C; after thecomplete reaction of the first-stage PCR, 4 μl of the pre-selectedarbitrary primer JYC4 (10 μM) corresponding to the 5′-end portion of theACPs are added to the reaction mixture and then the second stage PCRamplification is conducted as follows: 40 cycles of 94° C. for 40 sec,68° C. for 40 sec, and 72° C. for 40 sec; followed by a 5 min finalextension at 72° C.

[0611] Amplification products were resolved and analyzed byelectrophoresis in a 2.0 agarose gel which was stained with ethidiumbromide and photographed. The resulting PCR products can be alsodetected on a denaturing polyacrylamide gel by autoradiography ornon-radioactive detection methods such as silver staining (Gottschlichet al., 1997; Kociok et al., 1998), the use of fluoresenscent-labelledoligonucleotides (Bauer et al. 1993; Ito et al. 1994; Luehrsen et al.,1997; Smith et al., 1997), and the use of biotinylated primers (Korn etal., 1992; Tagle et al., 1993; Rosok et al., 1996).

[0612]FIG. 19 shows the results of an experiment in which a pair ofarbitrary ACPs were used to amplify segments of genomic DNA from avariety of mouse strains. To examine the reproducibility of genomicfingerprinting, the fingerprinting of each mouse strain was duplicatedusing two different sets of ACPs. The ACP -based PCR amplificationproduced several DNA segments from each set of primers and the resultswere reproducible. The polymorphisms were apparent between mice stains,indicating that mice stains can be distinguished through polymorphismsin genomic fingerprintings generated by ACP-based arbitrarily primedPCR. Thus, the ACP of the subject invention is useful to detectpolymorphisms and construct genetic maps.

EXAMPLE 8 Multiplex PCR Using ACP-based PCR

[0613] To demonstrate the application of ACP in multiplex PCR, theportions containing single nucleotide polymorphisms of human leukocyteadhesion molecule I (ELAM1) and human p53 (TP53) genes were amplifiedwith either conventional primers or ACP. The process and results for themultiplex PCR amplification using ACPs are described herein. DNAtemplate was obtained from human placenta.

[0614] The conventional primers for exon3 of ELAM1 (155 bp) used in theExample are: ELAM1N1 5′-TTGCACACTGTTGATTCTAA-3′; and (SEQ ID NO:67)ELAM1C1 5′-TTATTGATGGTCTCTACACA-3′. (SEQ ID NO:68)

[0615] The conventional primers for exon10 of ELAM1 (287 bp) used in theExample are: ELAM1N2 5′-CCACTGAGTCCAACATTC-3′; and (SEQ ID NO:69)ELAM1C2 5′-CTGAAACACTTCCCACAC-3′. (SEQ ID NO:70)

[0616] The conventional primers for exon4 of TP53 (349 bp) used in theExample are: P53N1 5′-CCTCTGACTGCTCTTTTCAC-3′; and (SEQ ID NO:71) P53C15′-ATTGAAGTCTCATGGAAGCC-3′. (SEQ ID NO:72)

[0617] The conventional primers for exons7-8 of TP53 (750 bp) used inthe Example are: P53N2 5′-TGCTTGCCACAGGTCTC-3′; and (SEQ ID NO:73) P53C25′-GCAGTGCTAGGAAAGAGG-3′. (SEQ ID NO:74)

[0618] These conventional primers used in the Example are known as theprimers that generate non-specific products in conventional mutiplex PCRmethods as known in the art.

[0619] The ACPs of the subject invention were applied to these fourconventional primer sets to demonstrate if the ACP can overcome theproblems such as non-specific products resulting from the use of theseconventional primer sets for multiplex PCR.

[0620] The 3′-end portions of the ACPs comprise the sequences of theabove conventional primers as follows and thus the size of ACPs is 26 bpor 27 bp bigger than that of the conventional primers: ELAM1N1-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIITTGCACACTGTTTGATTCTAA-3′; (SEQ ID NO:75)ELAM1C1-ACP 5′-TCACAGAAGTATGCCAAGCGAIIIIITTATTGATGGTCTCTACACA-3′; (SEQID NO:76) ELAM1N2-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIICCACTGAGTCCAACATTC-3′; (SEQ ID NO:77)ELAM1C2-ACP 5′-TCACAGAAGTATGCCAAGCGAIIIIICTGAAACACTTCCCACAC-3′; (SEQ IDNO:78) P53N1-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIICCTCTGACTGCTCTTTTCAC-3′;(SEQ ID NO:79) P53C1-ACP5′-TCACAGAAGTATGCCAAGCGAIIIIIATTGAAGTCTCATGGAAGCC-3′; (SEQ ID NO:80)P53N2-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIITGCTTGCCACAGGTCTC-3′; and (SEQID NO:81) P53C2-ACP 5′-TCACAGAAGTATGCCAAGCGAIIIIIGCAGTGCTAGGAAAGAGG-3′.(SEQ ID NO:82)

[0621] The 5′-end portion sequences of the ACPs comprise and serve aspre-selected arbitrary primer sequences only for the second-stage PCRamplification: JYC3 5′-TCACAGAAGTATGCCAAGCGA-3′ and (SEQ ID NO:11) JYC45′-GTCTACCAGGCATTCGCTTCAT-3′. (SEQ ID NO:12)

[0622] Multiplex PCR amplifications were conducted by one-stop ornon-stop two-stage PCR amplifications, which is a unique feature of thepresent invention. The PCR amplification was performed by hot start PCRmethod as described in Example 2.

[0623] PROTOCOL A: One-stop two-stage PCR amplifications

[0624] (A) First-stage PCR Amplification

[0625] The first-stage PCR amplification was conducted by two cycles ofPCR comprising of annealing, extending and denaturing reaction; thereaction mixture in the final volume of 49.5 μl containing 50 ng ofhuman genomic DNA, 8 μl of 10× PCR reaction buffer (Promega), 7 μl of 25mM MgCl₂, 5 μl of dNTP (2 mM each dATP, dCTP, dGTP, dTTP), each 0.5 μlof each 5′ ACP (10 μM) and 3′ ACP (10 μM) set is pre-heated at 94° C.,while holding the tube containing the reaction mixture at the 94° C.,0.5 μl of Taq polymerase (5 units/μl; Promega) is added into thereaction mixture; the PCR reactions are as follows: two cycles of 94° C.for 40 sec, 60° C. for 40 sec, and 72° C. for 40 sec; followed bydenaturing the amplification product at 94° C.

[0626] (B) Second-stage PCR amplification

[0627] The resultant products generated by the first-stage PCRamplification using multiple sets of the ACPs were then amplified by thefollowing second-stage PCR amplification under higher annealingtemperature. After the completion of the first-stage PCR amplification,each 2 ∥l of 10 μM pre-selected arbitrary primers, JYC3 and JYC4, wasadded into the reaction mixture obtained from the first-stage PCRamplification, under denaturing temperature such as at 94° C. The secondstage-PCR reaction was as follows: 40 cycles of 94° C. for 40 sec, 68°C. for 40 sec, and 72° C. for 1 min; followed by a 5 min final extensionat 72° C.

[0628] The amplified products were analyzed by electrophoresis in a 2%agarose gel and detected by staining with ethidium bromide. Theresulting PCR products can be also detected on a denaturingpolyacrylamide gel by autoradiography or non-radioactive detectionmethods such as silver staining (Gottschlich et al., 1997; Kociok etal., 1998), the use of fluoresenscent-labelled oligonucleotides (Baueret al. 1993; Ito et al. 1994; Luehrsen et al., 1997; Smith et al.,1997), and the use of biotinylated primers (torn et al., 1992; Tagle etal., 1993; Rosok et al., 1996).

[0629]FIGS. 20 and 21 show the results of experiments in which three orfour sets of primers were used to amplify mutiplex segments of genomicDNA at one reaction. The conventional primer sets generated non-specificproducts as well as specific-target products from three sets (FIG. 20A)or four sets (FIG. 21A) of primers. In contrast, the three sets (FIG.20B) or four sets (FIG. 21B) of ACPs produced only mutiplex targetproducts. Thus, the ACP of the subject invention can be used for theapplication of multiplex PCR.

[0630] PROTOCOL B: Non-stop two-stage PCR amplifications

[0631] Alternatively, the complete sequences of each ACP set, instead ofthe pre-selected arbitrary primers such as JYC3 and JYC4, can be used asprimers for the second-stage PCR amplification at the high stringentconditions. In this case, it is not necessary to add the pre-selectedarbitrary primers to the reaction mixture at the time of or after thefirst-stage PCR reaction.

[0632] The process of the non-stop two-stage PCR amplifications isbasically identical to Protocol A, except that the second stage PCRamplification should immediately follow first stage PCR amplificationwithout any delay because there is no step of adding pre-selectedarbitrary primers and that the concentration of each ACP set, each 1 μlof 5′ ACP (10 μM) and 3′ ACP (10 μM) set, is added at the first stagePCR amplification.

[0633] The amplified products were analyzed by electrophoresis in a 2%agarose gel and detected by staining with ethidium bromide. Theresulting PCR products can be also detected on a denaturingpolyacrylamide gel by autoradiography or non-radioactive detectionmethods such as silver staining (Gottschlich et al., 1997; Kociok etal., 1998), the use of fluoresenscent-labelled oligonucleotides (Baueret al. 1993; Ito et al. 1994; Luehrsen et al., 1997; Smith et al.,1997), and the use of biotinylated primers (Korn et al., 1992; Tagle etal., 1993; Rosok et al., 1996).

[0634] Consistent with the results of one-stop two-stage PCRamplifications (FIG. 21B), non-stop two-stage PCR amplification alsoproduced only target multiplex specific products (FIG. 21C). Theseexamples illustrate that ACP permits the products to be free from thebackground problems as well as non-specificity arising from theconventional primers used in multiplex PCR methods as known in the art.

EXAMPLE 9 Identification of Conserved Homology Segments in MultigeneFamilies Using ACP

[0635] The ACP of the subject invention was applied to detect and cloneconserved homology segments in multigene families. In the presentexample, degenerate primers were designed to detect homeobox sequences.The homeobox genes are characterized by a conserved 180-bp nucleotidesequence known as the homeobox, which encodes a 60-aa DNA bindinghomeodomain. To isolate homeobox genes involved in mouse embryodevelopment, total RNA obtained from three different stages of conceptusdevelopment, mouse 4.5-, 11.5-, and 18.5-day-old conceptuses, was usedas a starting material. First-strand cDNAs were prepared under the sameconditions as used in the cDNA synthesis of Example 3, whereinJYC5-T₁₅-ACP was used as the first-strand cDNA synthesis primer.

[0636] The following ACPs comprise the degenerate sequences for homeoboxsequence at their 3′-end portions and were used as degeneratehomeobox-specific primers for the first-stage PCR amplification:JYC2-HD1 (SEQ ID NO:83) 5′-GCTTGACTACGATACTGTGCGAIIIIIGTNCRRGTGTGGTT-3′;JYC2-HD2 (SEQ ID NO:84) 5′-GCTTGACTACGATACTGTGCGAIIIIIGTNCRRGTCTGGTT-3′;and JYC2-HD3 (SEQ ID NO:85)5′-GCTTGACTACGATACTGTGCGAIIIIIGTNCRRGTTTGGTT-3′.

[0637] The PCR amplification was performed by hot start PCR method asdescribed in Example 2 and conducted by one-stop or non-stop two-stagePCR amplifications. The following is an example of the process ofone-stop two-stage PCR amplifications.

[0638] 1. Combine the following reagents in a sterile 0.2 mlmicrocentrifuge tube: 49.5 μl of the total volume containing 1 μl offirst-strand cDNA (50 ng/μl), 5 μl of 10× PCR buffer (Roche), 5 μl of 2mM dNTP, 1 μl of one of 10 μM JYC2-HD1, JYC2-HD2, or JYC2-HD3 (5′primer), 1 μl of 10 μM JYC5-T₁₅-ACP (3′ primer) and 36.5 μl of steriledH₂O.

[0639] 2. Mix contents and spin the tube briefly in a microcentrifuge.

[0640] 3. Place the tube in the preheated thermal cycler at 94° C.

[0641] 4. Add 0.5 μl of Taq polymerase (5 units/μl; Roche) into thereaction while holding the tube at the temperature 94° C.

[0642] 5. Conduct PCR reaction under the following conditions: one cycleof 94° C. for 1 min, 52° C. for 3 min, and 72° C. for 1 min; followed by40 cycles of 94° C. for 40 sec, 65° C. for 40 sec, and 72° C. for 40sec; and followed by a 5 min final extension at 72° C.

[0643] The amplified products were analyzed by electrophoresis in a 2%agarose gel and detected by staining with ethidium bromide. Theresulting PCR products can be also detected on a denaturingpolyacrylamide gel by autoradiography or non-radioactive detectionmethods such as silver staining (Gottschlich et al., 1997; Kociok etal., 1998), the use of fluoresenscent-labelled oligonucleotides (Baueret al. 1993; Ito et al. 1994; Luehrsen et al., 1997; Smith et al.,1997), and the use of biotinylated primers (Korn et al., 1992; Tagle etal., 1993; Rosok et al., 1996).

[0644] Many differentially expressed bands in a specific stage wereobtained, subcloned into the pGEM-T Easy vector (Promega), andsequenced. Sequence analysis reveals that some of the clones containhomeobox sequences. Northern blot or RT-PCR analysis shows that theclones are identical to the results of the expression patterns observedby the electrophoresis. These results indicate that the method using theACP of the present invention for isolating conserved homology segmentsin multigene families produces only real PCR products. Freedom fromfalse positives, which is one major bottleneck remaining in the previousPCR-based techniques for isolating conserved homology segments inmultigene families, allows avoiding the subsequent labor-intensive workrequired for the verification of the amplified cDNA fragments.

EXAMPLE 10 Single Nucleotide Polymorphism Genotyping Using ACP-based PCR

[0645] To demonstrate the application of ACP in single nucleotidepolymorphism genotyping, a portion containing a single nucleotidepolymorphism (SNP) of human p53 (TP53) gene was amplified with eitherconventional primer or ACP. The process and results for the SNPgenotyping using ACPs are described herein. DNA templates were obtainedfrom human blood samples which have a SNP in exon 4 of the TP53 gene.This polymorphism is expressed as an Arg→Pro substitution at amino acidposition 72 by replacing G with C. A 349 nt sequence between nucleotide11991 and 12339 of the TP53 gene was amplified from each type oftemplate by a set of the following primers: P53N (SEQ ID NO:86)5′-CCTCTGACTGCTCTTTTCAC-3′ and P53C-ACP (SEQ ID NO:87)5′-TCACAGAAGTATGCCAAGCGAIIIIIATTGAAGTCTCATGGAAGCC- 3′.

[0646] The amplified products containing the SNP between their ends wereused as templates for detecting the SNP using allele-specific ACPs asfollows: P53N1A-ACP (SEQ ID NO:88) 5′-GTCTACCAGCATTCGCTTCATIIIIICCCC GCGTGG-3′, P53N1B-ACP (SEQ ID NO:89) 5′-GTCTACCAGGCATTCGCTTCATIIIIICCCC CCGTGG-3′, P53N2A-ACP (SEQ ID NO:90) 5′-GTCTACCAGGCATTCGCTTCATIIIIITCCCCG CGTG-3′, P53N2B-ACP (SEQ ID NO:91) 5′-GTCTACCAGGCATTCGCTTCATIIIIITCCCCC CGTG-3′, P53N3A-ACP (SEQ ID NO:92)5′-GTCTACCAGGCATTCGCTTCATIIIIICTCCCC G CGT-3′, P53N3B-ACP (SEQ ID NO:93)5′-GTCTACCAGGCATTCGCTITCATIIIIICTCCCC C CGT-3′, P53N4A-ACP (SEQ IDNO:94) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC G CG-3′, P53N4B-ACP (SEQ IDNO:95) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC C CG-3′, P53N5A-ACP (SEQ IDNO:96) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC G -3′, and P53N5B-ACP (SEQID NO:97) 5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC C -3′.

[0647] The polymorphic base is underlined at the 3′-end portion of eachallele-specific ACP and the position of the polymorphic base isconsidered an interrogation position. The interrogation position isplaced at several different positions from the 3′-end of allele-specificACPs in order to determine the most critical position in annealingspecificity for detecting the SNP.

[0648] The allele-specific ACPs were used as 5′ primers. P53C-ACP andone of P53N1A-ACP, P53N2A-ACP, P53N3A-ACP, P53N4A-ACP and P53N5A-ACPwere used for wild-type A genotyping. P53C-ACP and one of P53N1B-ACP,P53N2B-ACP, P53N3B-ACP, P53N4B-ACP and P53N5B-ACP were used forvariant-type B genotyping. The 5′-end portion sequences of the ACPs wereserved as pre-selected arbitrary primer sequences for the second-stagePCR amplification: JYC3 5′-TCACAGAAGTATGCCAAGCGA-3′ and (SEQ ID NO:11)JYC4 5′-GTCTACCAGGCATTCGCTTCAT-3′. (SEQ ID NO:12)

[0649] (A) First-stage PCR Amplification

[0650] The first-stage PCR amplification was conducted by one cycle ofPCR consisting of annealing, extending and denaturing reaction; thereaction mixture in a final volume of 49.5 μl containing 1 μl of theamplified target genomic segment containing the SNP in exon 4 of theTP53 gene, 5 μl of 10× PCR reaction buffer (Promega), 5 μl of 25 mMMgCl₂, 5 μl of dNTP (2 mM each dATP, dCTP, dGTP, dTTP), and 1 μl of oneof allele-specific ACPs (10 μM) is pre-heated at 94° C., while holdingthe tube containing the reaction mixture at the 94° C., 0.5 μl of Taqpolymerase (5 units/μl; Promega) is added into the reaction mixture; forthe allele-specific ACPs having 10 nucleotides at its 3′-end portion,the PCR reactions are as follows: one cycle of 94° C. for 40 sec, 55° C.for 40 sec, and 72° C. for 40 sec; followed by denaturing theamplification product at 94° C; for the allele-specific ACPs having 8nucleotides at its 3′-end portion, the PCR reactions are as follows: onecycle of 94° C. for 40 sec, 50° C. for 40 sec, and 72° C. for 40 sec;followed by denaturing the amplification product at 94° C. The resultantproduct is a first DNA strand complementary to the target genomicsegment.

[0651] (B) Second-stage PCR Amplification

[0652] The resultant product generated by the first-stage PCRamplification was then amplified by the following second-stage PCRamplification at a higher annealing temperature than the first annealingtemperature. After the completion of the first-stage PCR amplification,1 μl of 10 μM pre-selected arbitrary primer, JYC3, was added into thereaction mixture obtained from the first-stage PCR amplification, underdenaturing temperature such as at 94° C. The second stage-PCR reactionwas performed as follows: 30 cycles of 94° C. for 40 sec, 68° C. for 40sec, and 72° C. for 40 sec; followed by a 5 min final extension at 72°C.

[0653] The amplified products were analyzed by electrophoresis in a 2%agarose gel and detected by staining with ethidium bromide. Theresulting PCR products can be also detected on a denaturingpolyacrylamide gel by autoradiography or non-radioactive detectionmethods such as silver staining (Gottschlich et al., 1997; Kociok etal., 1998), the use of fluoresenscent-labelled oligonucleotides (Baueret al. 1993; Ito et al. 1994; Luehrsen et al., 1997; Smith et al.,1997), and the use of biotinylated primers (Korn et al., 1992; Tagle etal., 1993; Rosok et al., 1996).

[0654]FIG. 22 shows the results of allele-specific amplification usingACP. The pair of wild-type A-specific ACPs (P53N2A-ACP and P53C-ACP)generated a specific target product only from the samples havinghomozygous wild-type A (lane 1) or heterozygous genotyping (lane 3), butnot from the samples having homozygous variant-type B genotyping (lane5). The pair of variant-type B-specific ACPs (P53N2B-ACP and P53C-ACP)generated a specific target product only from the samples havinghomozygous variant-type B (lane 6) or heterozygous genotyping (lane 4),but not from the samples having homozygous wild-type A genotyping (lane2). These results indicate that the ACP of the subject invention can beapplied as an easy and economic method for detecting the genotype ofSNPs since the use of fluorescent DNA probe nor post-PCR processing isnot required in this approach. The allele-specific ACPs each having aninterrogation position at its 3′-end portion showed improvement ofannealing specificity. Moreover, when the allele-specific ACP has aninterrogation position at the position 5 from the 3′-end (e.g.,P53N2A-ACP and P53N2B-ACP), the annealing specificity is most criticallyaccomplished.

[0655] In order to verify if the position 5 from the actual 3′-end ofthe allele-specific ACP is the most appropriate for the interrogationposition, additional six experiments were conducted using the sameprocess as used in FIG. 22. DNA templates were obtained from human bloodsamples which have a SNP. Six short genomic fragments containing SNPswere amplified using each different primer set as follows:

[0656] 703N 5′-ATTCTGATGGTGTGGATTGTG-3′(SEQ ID NO:98) and SM703C5′-TCACAGAAGTATGCCAAGCGAIIIIIRACCCTGGAGTAGACGAAGA-3′ (SEQ ID NO:99) forBeta-2 adrenergic receptor (ADRB2),

[0657] 028N 5′-CCTTCTGTGCTTGATGCTTTT-3′(SEQ ID NO:102) and SM028C5′-TCACAGAAGTATGCCAAGCGAIIIIICAGGAAGGATGAGCATTTAG-3′(SEQ ID NO:103) forChemokine (c-c motif) receptor 5 (CCR5),

[0658] 695N: 5′-AGAAAAACCAGAGGCAGCTT-3′(SEQ ID NO: 106) and SM695C5′-TCACAGAAGTATGCCAAGCGAIIIIIAGCACAAACCAAAGACACAGT-3′ (SEQ ID NO: 107)for Interleukin 13 receptor,

[0659] 679N 5′-CTAGCTGCAAGTGACATCTCT-3′(SEQ ID NO: 110) and SM679C5′-TCACAGAGTATCCAAGCGIIIIITCAGTAAGAAGCCAGGAGAG-3′(SEQ ID NO: 111) forLeukocyte adhesion molecule-1 (LAM-1),

[0660] 832N 5′-TTTTGGGTGGAGGCTAACAT-3′(SEQ ID NO: 114) and

[0661] SM832C: 5′-TCACAGAAGTATGCCAGCGAIIIIIAACGATGCAGACACCACCA-3′(SEQ IDNO: 115) for Tachykinin receptor 3 (TACR3), and

[0662] 880N 5′-CTTCCACCAATACTCTTTTCC-3′(SEQ ID NO:118) and

[0663] SM880C : 5′-TCACAGAAGTATGCCAGCGAIIIII GCATACACACAAGAGGCAGA-3′(SEQ ID NO: 119) for Interleukin 1, beta (IL1B).

[0664] The amplified products containing the SNP between their ends wereused as templates for detecting the SNPs wherein allele-specific ACPswere applied as follows:

[0665] SM703 -A 5′-GTCTACCAGGCATTCGCTTCATIIIIIGGTACAGGGC-3′(SEQ ID NO:100) and SM703-B 5′-GTCTACCAGGCATTCGCTTCATIIIIIGGTACCGGGC-3′(SEQ ID NO:101) for Beta-2 adrenergic receptor (ADRB2),

[0666] SM028-A 5′-GTCTACCAGGCATTCGCTTCATIIIIITCCAAACCAA-3′(SEQ IDNO:104) and SM028-B 5′-GTCTACCAGGCATTCGCTTCATIIIIITCCAACCCAA-3′(SEQ IDNO:105) for Chemokine (c-c motif) receptor 5 (CCR5),

[0667] SM695-A 5′-GTCTACCAGGCATTCGCTTCATIIIII CCATTTTAGG-3′(SEQ ID NO:108) and SM695-B 5′-GTCTACCAGGCATTCGCTTCATIIIII CCATTGTAGG-3′(SEQ ID NO:109) for Interleukin 13 receptor,

[0668] SM679-A 5′-GTCTACCAGGCATTCGCTTCATIIIIICCAGAACTTT-3′(SEQ ID NO:112) and SM679-B 5′-GTCTACCAGGCATTCGCTTCATIIIIICCAGACCTTT-3′(SEQ IDNO:113) for Leukocyte adhesion molecule-1 (LAM-1),

[0669] SM832-A 5′-GTCTACCAGGCATTCGCTTCATIIIIIGACTGGTAAA-3′(SEQ ID NO:116) and SM832-B 5′-GTCTACCAGGCATTCGCTTCATIIIIIGACTGATAAA-3′(SEQ ID NO:117) for Tachykinin receptor 3 (TACR3), and

[0670] SM880-A 5′-GTCTACCAGGCATTCGCTTCATIIIIIAAAGCCATAA-3′(SEQ ID NO:120) and SM880-B 5′-GTCTACCAGGCATTCGCTTCATIIIIIAAAGCTATAA-3′(SEQ IDNO:121) for Interleukin 1, beta (IL1B).

[0671]FIG. 23 shows the results of allele-specific amplifications forsix additional SNPs each present in different gene such as Beta-2adrenergic receptor (ADRB2) (A), Chemokine (c-c motif) receptor 5 (CCR5)(B), Interleukin 13 receptor (C), Leukocyte adhesion molecule-1 (LAM-1)(D), Tachykinin receptor 3 (TACR3) (E), and Interleukin 1, beta (IL1B)(F). Consistant with the results of FIG. 22, the annealing specificityis critically accomplished when the allele-specific ACP has aninterrogation position at the position 5 from the 3′-end. The pair ofwild-type A-specific ACPs generated a specific target product only fromthe samples having homozygous wild-type A (lane 1) or heterozygousgenotyping (lane 3), but not from the samples having homozygousvariant-type B genotyping (lane 5). The pair of variant-type B-specificACPs generated a specific target product only from the samples havinghomozygous variant-type B (lane 6) or heterozygous genotyping (lane 4),but not from the samples having homozygous wild-type A genotyping (lane2).

[0672] Having described a preferred embodiment of the present invention,it is to be understood that variants and modifications thereof fallingwithin the spirit of the invention may become apparent to those skilledin this art, and the scope of this invention is to be determined byappended claims and their equivalents. TABLE 1 SEQ ID NO DesignationSequence Information 1 ACP1 5′-GTCTACCAGGCATTCGCTTCATIIIIICAGGAGTGG-3′ 2ACP2 5′-GTCTACCAGGCATTCGCTTCATIIIIIGGCGACGATS-3′ 3 ACP35′-GTCTACCAGGCATTCGCTTCATIIIIIGCCATCGACS-3′ 4 ACP45′-GTCTACCAGGCATTCGCTTCATIIIIIAGATGCCCGW-3′ 5 ACP55′-GTCTACCAGGCATTCGCTTCATTIIIIIAGGCGATGCS-3′ 6 ACP65′-GTCTACCAGGCATTCGCTTCATIIIIITCTCCCGGTS-3′ 7 ACP75′-GTCTACCAGGCATTCGCTTCATIIIIITTGTGGCGGS-3′ 8 ACP85′-GTCTACCAGGCATTCGCTTCATIIIIICTCCGATGCS-3′ 9 ACP95′-GTCTACCAGGCATTCGCTTCATIIIIICCTGCGGGTW-3′ 10 JYC25′-GCTTGACTACGATACTGTGCGA-3′ 11 JYC3 5′-TCACAGAAGTATGCCAAGCGA-3′ 12 JYC45′-GTCTACCAGGCATTCGCTTCAT-3′ 13 ACP105′-GTCTACCAGGCATTCGCTTCATIIIIIGCCATCGACC-3′ 14 ACP115′-GTCTACCAGGCATTCGCTTCATIIIIIGCCATCGACG-3′ 15 ACP125′-GTCTACCAGGCATTCGCTTCATIIIIIAGGCGATGCC-3′ 16 ACP135′-GTCTACCAGGCATTCGCTTCATIIIIIAGGCGATGCG-3′ 17 ACP145′-GTCTACCAGGCATTCGCTTCATIIIIICTCCGATGCC-3′ 18 ACP155′-GTCTACCAGGCATTCGCTTCATIIIIICTCCGATGCG-3′ 19 CRP2105′-GTCTACCAGGCATTCGCTTCATGCCATCGACC-3′ 20 ACP165′-GTCTACCAGGCATTCGCTTCATIIGCCATCGACC-3′ 21 ACP175′-GTCTACCAGGCATTCGCTTCATIIIIGCCATCGACC-3′ 22 ACP185′-GTCTACCAGGCATTCGCTTCATIIIIIIGCCATCGACC-3′ 23 ACP195′-GTCTACCAGGCATTCGCTTCATHIIIIIIGCCATCGACC-3′ 24 dT-JYC35′-CACAGAAGTATGCCAAGCGACTCGAGTTTTTTTTTTTTTTT-3′ 25 dT-JYC25′-GCTTGACTACGATACTGTGCGATTTTTTTTTTTTTTT-3′ 26 JYC2-T13C5′-CTTGACTACGATACTGTGCGATTTTTTTTTTTTTC-3′ 27 JYC2-T13G5′-GCTTGACTACGATACTGTGCGATTTTTTTTTTTTTG-3′ 28 JYC2-T13A5′-GCTTGACTACGATACTGTGCGATTTTTTTTTTTTTA-3′ 29 dT¹⁰⁻JYC25′-GCTTGACTACGATACTGTGCGATTTTTTTTTT-3′ 30 dT¹⁰⁻ACP15′-GCTTGACTACGATACTGTGCGAIIIIITTTTTTTTTT-3′ 31 DEG 2GCCATCGACCCGTTTCTCTAGCCCCATCTTCATGTGTTTTAATGAGATGATATTAATTCATTACATTCATGGATAATATGTCCCTGAGTACATTCTAATCTAGATTTAACTTCAAAAAAAAAAAAAAAAA 32 DEG 5AGGCGATGCGGGCTGTACTCTGGGTGGCTGCCACAGTCTCATGAGAAACCAAGGGCAAAGGACCAAGGAAAAGGGTCTCAGGCCCCTAAAGCAGTGGCTTTCAACCATCCTAATGTTGTGACCTTTTAATACAGTTCCTCATGTTGTGTGACCCCCCAACCATAAAATGATTTTTGTTTCTACTTCAAAAAAAAAAAAAAAAAAAAAAA 33 SMART IV5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCr(GGG)-3′ 34 5′ PCR Primer5′-AAGCAGTGGTATCAACGCAGAGT-3′ 35 CDS III/3′5′-ATTCTAGAGGCCGAGGCGGCCGACATG-(dT)₃₀VN-3′ 36 rG3-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIIGGr(GGG)-3′ 37 rG2-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIIGGr(GG)d(G)-3′ 38 dG3-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIIGGd(GGG)-3′ 39 Oligo dT¹⁸⁻ACP5′-GCTTGACTACGATACTGTGCGAIIIIITTTTTTTTTTTTTTTTTT-3′ 40 PLP-Cα5′-GAGAGGATAGTTTCAGGGAC-3′ 41 JunB3 5′-CTCCGTGGTACGCCTGCTTTCTC-3′ 42β-actin 1 5′-TCGTCACCCACATAGGAGTC-3′ 43 β-actin 25′-CTAAGAGGAGGATGGTCGC-3′ 44 EsxN7 5′-GCCGGTTGCAGAGCACC-3′ 45 EsxC65′-GAACCATGTTTCTGAATGCC-3′ 46 EsxN7-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIIGCCGGTTGCAGAGCACG-3′ 47 EsxC6-ACP5′-GCTTGACTACGATACTGTGCGAIIIIIGAACCATGTTTCTGAATGCC-3′ 48 EsxN15′-GAATCTGAAACAACTTTCTA-3′ 49 EsxC2 5′-GATGCATGGGACGAGGCACC-3′ 50EsxN1-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIIGAATCTGAAACAACTTTCTA-3′ 51 EsxN35′-CGCCGCACCCCTGCCCGCA-3′ 52 EsxC5 5′-GATGCATGGGACGAGGCA-3′ 53 EsxN3-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIICGCCGCACCCCTGCCCGCA-3′ 54 Oligo-dT₁₅5′-TTTTTTTTTTTTTTT-3′ 55 EsxC2-ACP5′-GCTTGACTACGATACTGTGCGAIIIIIGATGCATGGGACGAGGCACC-3′ 56 EsxC5-ACP5′-GCTTGACTACGATACTGTGCGAIIIIIGATGCATGGGACGAGGCA-3′ 57 OligoVdT¹⁵⁻ACP5′-GCTTGACTACGATACTGTGCGAIIIIITTTTTTTTTTTTTTTV-3′ 58 dN⁶⁻ACP5′-GCTTGACTACGATACTGTGCGAIIIIINNNNN-3′ 59 rG1-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIIGGr(G)d(GG)-3′ 60 JYC55′-CTGTGAATGCTGCGACTACGAT-3′ 61 JYC5-T¹⁵⁻ACP5′-CTGTGAATGCTGCGACTACGATIIIIITTTTTTTTTTTTTTT-3′ 62 JYC5-T₁₅V-ACP5′-CTGTGAATGCTGCGACTACGATIIIIITTTTTTTTTTTTTTV-3′ 63 JYC5-T₁₅VN-ACP5′-CTGTGAATGCTGCGACTACGATIIIIITTTTTTTTTTTTTTTVN-3′ 64 ACP1015′-GTCTACCAGGCATTCGCTTCATIIIIICCGGAGGATC-3′ 65 ACP1095′-GTCTACCAGGCATFFCGCTTCATIIIIICTGCAGGACG-3′ 66 ACP1165′-GTCTACCAGGCATTCGCTTCATIIIIICGGAGCATCC-3′ 67 ELAM1N15′-TTGCACACTGTTGATTCTAA-3′ 68 ELAM1C1 5′-TTATTGATGGTCTCTACACA-3′ 69ELAM1N2 5′-CCACTGAGTCCAACATTC-3′ 70 ELAM1C2 5′-CTGAAACACTTCCCACAC-3′ 71P53N1 5′-CCTCTGACTGCTCTTTTCAC-3′ 72 P53C1 5′-ATTGAAGTCTCATGGAAGCC-3′ 73P53N2 5′-TGCTTGCCACAGGTCTC-3′ 74 P53C2 5′-GCAGTGCTAGGAAAGAGG-3′ 75ELAM1N1-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIITTGCACACTGTTGATTCTAA-3′ 76ELAM1C1-ACP 5′-TCACAGAAGTATGCCAAGCGAIIIIITTATTGATGGTCTCTACACA-3′ 77ELAM1N2-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIICCACTGAGTCCAACATTC-3′ 78ELAM1C2-ACP 5′-TCACAGAAGTATGCCAAGCGAIIIIICTGAAACACTTCCCACAC-3′ 79P53N1-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIICCTCTGACTGCTCTTTTCAC-3′ 80P53C1-ACP 5′-TCACAGAAGTATGCCAAGCGAIIIIIATTGAAGTCTCATGGAAGCC-3′ 81P53N2-ACP 5′-GTCTACCAGGCATTCGCTTCATIIIIITGCTTGCCACAGGTCTC-3′ 82P53C2-ACP 5′-TCACAGAAGTATGCCAAGCGAIIIIIGCAGTGCTAGGAAAGAGG-3′ 83 JYC2-HD15′-GCTTGACTACGATACTGTGCGAIIIIIGTNCRRGTGTGGTT-3′ 84 JYC2-HD25′-GCTTGACTACGATACTGTGCGAIIIIIGTNCRRGTCTGGTT-3′ 85 JYC2-HD35′-GCTTGACTACGATACTGTGCGAIIIIIGTNCRRGTTTGGTT-3′ 86 P53N5′-CCTCTGACTGCTCTTTTCAC-3′ 87 P53C-ACP5′-TCACAGAAGTATGCCAAGCGAIIIIIATTGAAGTCTCATGGAAGCC-3′ 88 PS3N1A-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIICCCC G CGTGG-3′ 89 PS3N1B-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIICCCC C CGTGG-3′ 90 P53N2A-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIITCCCC G CGTG-3′ 91 PS3N2B-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIITCCCC C CGTG-3′ 92 PS3N3A-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIICTCCCC G CGT-3′ 93 P53N3B-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIICTCCCC C CGT-3′ 94 P53N4A-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC G CG-3′ 95 P53N4B-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC C CG-3′ 96 P53N5A-ACP5′-GTCTACCAGGCATTCGCTTCATIIIITGCTCCCC G -3′ 97 P53NSB-ACP5′-GTCTACCAGGCATTCGCTTCATIIIIIGCTCCCC C -3′ 98 703N5′-ATTCTGATGGTGTGGATTGTG-3′ 99 SM703C5′-TCACAGAAGTATGCCAAGCGAIIIIIACCCTGGAGTAGACGAAGA-3′ 100 SM703-A5′-GTCTACCAGGCATTCGCCTTCATIIIIIGGTAC A GGGC-3′ 101 SM703-B5′-GTCTACCAGGCATTCGCTTCATIIIIIGGTAC C GGGC-3′ 102 028N5′-CCTTCTGTGCTTGATGCTTTT-3′ 103 SM028C5′-TCACAGAAGTATGCCAAGCGAIIIIICAGGAAGGATGAGCATTTAG-3′ 104 SM028-A5′-GTCTACCAGGCATTCGCTTCATIIIIITCCAA A CCAA-3′ 105 SM028-B5′-GTCTACCAGGCATTCGCTTCATIIIIITCCAA C CCAA-3′ 106 695N5′-AGAAAAACCAGAGGCAGCTT-3′ 107 SM69SC5′-TCACAGAAGTATGCCAAGCGAIIIIIAGCACAAACCAAAGACACAGT-3′ 108 SM695-A5′-GTCTACCAGGCATTCGCTTCATIIIIICCATT T TAGG-3′ 109 SM695-B5′-GTCTACCAGGCATTCGCTTCATIIIIICCATT G TAGG-3′ 110 679N5′-CTAGCTGCAAGTGACATCTCT-3′ 111 SM679C5′-TCACAGAGTATCCAAGCGIIIIITCAGTAAGAAGCCAGGAGAG-3′ 112 SM679-A5′-GTCTACCAGGCATTCGCTTCATIIIIICCAGA A CTTT-3′ 113 SM679-B5′-GTCTACCAGGCATTCGCTTCATIIIIICCAGA C CTTT-3′ 114 832N5′-TTTTGGGTGGAGGCTAACAT-3′ 115 SM832C5′-TCACAGAAGTATGCCAGCGAIIIIIAACGATGCAGACACCACCA-3′ 116 SM832-A5′-GTCTACCAGGCATTCGCTTCATIIIIIIGACTG G TAAA-3′ 117 SM832-B5′-GTCTACCAGGCATTCGCTTCATIIIIIGACTG A TAAA-3′ 118 880N5′-CTTCCACCAATACTCTTTTCC-3′ 119 SM880C5′-TCACAGAAGTATGCCAGCGAIIIIIGCATACACACAAGAGGCAGA-3′ 120 SM880-A5′-GTCTACCAGGCATTCGCTTCATIIIIIAAAGC C ATAA-3′ 121 SM880-B5′-GTCTACCAGGCATTCGCTTCATIIIIIAAAGC T ATAA-3′

[0673] TABLE 2 Differentially Expressed cDNA Fragments Cloned by the ACPof the Present Invention Nomenclature Identity Homology DEG 1Tropomyosin 2 (beta) Mouse 92% DEG 2 Novel Novel DEG 3 Hypotheticalprotein (Tes gene) Mouse 99% DEG 4 Protease-6 Mouse 92% DEG 5 NovelNovel DEG 6 Cytoebrome c oxidase, subunit Vb Mouse 99% DEG 7Hydroxylacyl-Coenzyme A dehydrogense Mouse 98% (Hadh) DEG 8 Troponin T2,cardiac (Tnnt2) Mouse 94% DEG 9 RNA binding motif protein, X Mouse 96%chromosome DEG 10 Peroxiredoxin 6 (Prdx6) Mouse 89% DEG 11 11 days or 13days embryo cDNA Mouse 98%

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What is claimed is:
 1. An annealing control primer for improvingannealing specificity in nucleic acid amplification, which comprises:(a) a 3′-end portion having a hybridizing nucleotide sequencesubstantially complementary to a site on a template nucleic acid tohybridize therewith; (b) a 5′-end portion having a pre-selectedarbitrary nucleotide sequence; and (c) a regulator portion positionedbetween said 3′-end portion and said 5′-end portion comprising at leastone universal base or non-discriminatory base analog, whereby saidregulator portion is capable of regulating an annealing portion of saidprimer in association with annealing temperature.
 2. The annealingcontrol primer according to claim 1, wherein said pre-selected arbitrarynucleotide sequence of said 5′-end portion is substantially notcomplementary to any site on said template nucleic acid.
 3. Theannealing control primer according to claim 1, wherein said templatenucleic acid is gDNA, cDNA or mRNA.
 4. The annealing control primer ofclaim 3, wherein said gDNA or cDNA is single or double-stranded DNA. 5.The annealing control primer according to claim 1, wherein said nucleicacid amplification is performed under a first and a second annealingtemperatures.
 6. The annealing control primer according to claim 5,wherein said first annealing temperature in said nucleic acidamplification is identical to or lower than said second annealingtemperature.
 7. The annealing control primer according to claim 5,wherein said 3′-end portion is involved in annealing at said firstannealing temperature and said 5′-end portion serves as a priming siteat said second annealing temperature.
 8. The annealing control primeraccording to claim 5, wherein said regulator portion is capable ofrestricting said annealing portion of said primer to said 3′-end portionat said first annealing temperature.
 9. The annealing control primeraccording to claim 5, wherein said first annealing temperature isbetween about 30° C. and 68° C.
 10. The annealing control primeraccording to claim 5, wherein said second annealing temperature isbetween about 50° C. and 72° C.
 11. The annealing control primeraccording to claim 1, wherein said annealing control primer has ageneral formula of 5′-X_(p)-Y_(q)-Z_(r)-3′, wherein X_(p) representssaid 5′-end portion having said pre-selected arbitrary nucleotidesequence substantially not complementary to any site on the templatenucleic acid; Y_(q) represents said regulator portion comprising atleast one universal base or non-discriminatory base analog; Z_(r)represents said 3′-end portion having a hybridizing nucleotide sequencesubstantially complementary to a site on the template nucleic acid tohybridize therewith; wherein p, q and r represent the number ofnucleotides; and wherein X, Y and Z is deoxyribonucleotide orribonucleotide.
 12. The annealing control primer according to claim 11,wherein said universal base or non-discriminatory base analog is capableof forming base-pairs with each of the natural DNA/RNA bases with littlediscrimination between said natural DNA/RNA bases.
 13. The annealingcontrol primer according to claim 11, wherein said universal base ornon-discriminatory base analog is selected from the group consisting ofdeoxyinosine, inosine, 7-deaza-2′-deoxyinosine, 2-aza-2′-deoxyinosine,2′-OMe inosine, 2′-F inosine, deoxy 3-nitropyrrole, 3-nitropyrrole,2′-OMe 3-nitropyrrole, 2′-F 3-nitropyrrole,1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole, deoxy 5-nitroindole,5-nitroindole, 2′-OMe 5-nitroindole, 2′-F 5-nitroindole, deoxy4-nitrobenzimidazole, 4-nitrobenzimidazole, deoxy 4-aminobenzimidazole,4-aminobenzimidazole, deoxy nebularine, 2′-F nebularine, 2′-F4-nitrobenzimidazole, PNA-5-introindole, PNA-nebularine, PNA-inosine,PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole,morpholino-nebularine, morpholino-inosine,morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole,phosphoramidate-5-nitroindole, phosphoramidate-nebularine,phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole,phosphoramidate-3-nitropyrrole, 2′-0-methoxyethyl inosine,2′0-methoxyethyl nebularine, 2′-0-methoxyethyl 5-nitroindole,2′-O-methoxyethyl 4-nitro-benzimidazole, 2′-0-methoxyethyl3-nitropyrrole, and combinations thereof.
 14. The annealing controlprimer according to claim 11, wherein said universal base ornon-discriminatory base analog is deoxyinosine,1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole or 5-nitroindole. 15.The annealing control primer according to claim 11, wherein saidregulator portion comprises contiguous nucleotides having universal baseor non-discriminatory base analog.
 16. The annealing control primeraccording to claim 11, wherein said deoxyribonucleotide is naturallyoccurring dNMP, modified nucleotide and non-natural nucleotide.
 17. Theannealing control primer according to claim 11, wherein p represents aninteger of 15 to
 60. 18. The annealing control primer according to claim11, wherein q is at least
 2. 19. The annealing control primer accordingto claim 11, wherein q is at least
 3. 20. The annealing control primeraccording to claim 11, wherein q represents an integer of 2 to
 15. 21.The annealing control primer according to claim 11, wherein r representsan integer of 6 to
 50. 22. The annealing control primer according toclaim 11, wherein p is an integer of 15 to 60, q is an integer of 2 to15 and r is an integer of 6 to
 30. 23. The annealing control primeraccording to claim 11, wherein X_(p) comprises a universal primersequence.
 24. The annealing control primer according to claim 11,wherein X_(p) includes a sequence or sequences recognized by arestriction endonuclease or restriction endonucleases.
 25. The annealingcontrol primer according to claim 11, wherein X_(p) comprises at leastone nucleotide with a label for detection or isolation.
 26. Theannealing control primer according to claim 11, wherein Z_(r) is anucleotide sequence which hybridizes to the polyadenosine (polyA) tailof an mRNA.
 27. The annealing control primer according to claim 26,wherein Z_(r) comprises at least 10 contiguous deoxythymidinenucleotides.
 28. The annealing control primer according to claim 26,wherein Z_(r) comprises at least 10 contiguous deoxythymidinenucleotides having 3′-V at its 3′-end; in which V is one selected fromthe group consisting of deoxyadenosine, deoxycytidine anddeoxyguanosine.
 29. The annealing control primer according to claim 26,wherein Z_(r) comprises at least 10 contiguous deoxythymidinenucleotides having 3′-NV at its 3′-end; in which V is one selected fromthe group consisting of deoxyadenosine, deoxycytidine anddeoxyguanosine, and N is one selected from the group consisting ofdeoxyadenosine, deoxythymidine, deoxycytidine and deoxyguanosine. 30.The annealing control primer according to claim 11, wherein Z_(r) is anucleotide sequence substantially complementary to a target sequence inthe template nucleic acid.
 31. The annealing control primer according toclaim 11, wherein Z_(r) is a random nucleotide sequence.
 32. Theannealing control primer according to claim 11, wherein Z_(r) is anucleotide sequence substantially complementary to a consensus sequencefound in a gene family.
 33. The annealing control primer according toclaim 11, wherein Z_(r) is a degenerate nucleotide sequence selectedfrom a plurality of combinations of nucleotides encoding a predeterminedamino acid sequence.
 34. The annealing control primer according to claim11, wherein Z_(r) comprises at least one ribonucleotide.
 35. Theannealing control primer according to claim 11, wherein Z_(r) comprisesat least one nucleotide complementary to allelic site.
 36. The annealingcontrol primer according to claim 11, wherein Z_(r) comprises at leastone mismatch nucleotide to a target nucleic acid for mutagenesis.
 37. Akit comprising the primer or the primer set according to any one ofclaims 1-36.
 38. The kit according to claim 37, wherein the kit furthercomprises a primer or a primer pair having a nucleotide sequencecorresponding to the 5′-end portion of said primer.
 39. A method foramplifying a nucleic acid sequence from a DNA or a mixture of nucleicacids, wherein said method comprises performing an amplificationreaction using primers, characterized in that at least one primer hasthe same structure as the primer of claim
 1. 40. The method according toclaim 39, wherein said method is performed using two stageamplifications, which comprises: (a) performing a first-stageamplification of said nucleic acid sequence at a first annealingtemperature comprising at least two cycles of primer annealing, primerextending and denaturing, using the primer pair of claim leach having atits 3′-end portion a hybridizing sequence substantially complementary toa region of said nucleic acid sequence to hybridize therewith, underconditions in which each primer anneals to said region of said nucleicacid sequence, whereby the amplification product of said nucleic acidsequence is generated; and (b) performing a second-stage amplificationof said amplification product generated from step (a) at a secondannealing temperature, which is high stringent conditions, comprising atleast one cycle of primer annealing, primer extending and denaturing,using the same primers as used in step (a) or a primer pair eachcomprising a pre-selected arbitrary nucleotide sequence corresponding toeach 5′-end portion of said primers used in step (a), under conditionsin which each primer anneals to the 3′- and 5′-ends of saidamplification product, respectively, whereby said amplification productis re-amplified.
 41. A method for selectively amplifying a targetnucleic acid sequence from a DNA or a mixture of nucleic acids, whereinsaid method comprises performing an amplification reaction usingprimers, characterized in that at least one primer has the samestructure as the primer of claim
 1. 42. The method according to claim41, wherein said method is performed using two stage amplifications,which comprises: (a) performing a first-stage amplification of saidtarget nucleic acid sequence at a first annealing temperature comprisingat least two cycles of primer annealing, primer extending anddenaturing, using the primer pair of claim 1 each having at its 3′endportion a hybridizing sequence substantially complementary to a regionof said target nucleic acid sequence to hybridize therewith, underconditions in which each primer anneals to its target nucleotidesequence, whereby the amplification product of said target nucleotidesequence is generated; and (b) performing a second-stage amplificationof said amplification product generated from step (a) at a secondannealing temperature, which is high stringent conditions, comprising atleast one cycle of primer annealing, primer extending and denaturing,using the same primers as used in step (a) or a primer pair eachcomprising a pre-selected arbitrary nucleotide sequence corresponding toeach 5′-end portion of said primers used in step (a), under conditionsin which each primer anneals to the 3′- and 5′-ends of saidamplification product, respectively, whereby said amplification productis re-amplified.
 43. A method for selectively amplifying a targetnucleic acid sequence from an mRNA, wherein said method comprisesreverse transcribing said mRNA and performing an amplification reactionusing primers, characterized in that at least one primer has the samestructure as the primer of claim
 1. 44. The method according to claim43, wherein said method is performed using two stage amplifications,which comprises: (a) contacting said mRNA with an oligonucleotide dTprimer which is hybridized to polyA tail of said mRNA under conditionssufficient for template driven enzymatic deoxyribonucleic acid synthesisto occur; (b) reverse transcribing said mRNA to which saidoligonucloetide dT pirmer hybridizes to produce a first DNA strand thatis complementary to said mRNA to which said oligonucloetide dT pirmerhybridizes; (c) performing a first-stage amplification of said targetnucleic acid sequence from said first DNA strand obtained from step (b)at a first annealing temperature comprising at least two cycles ofprimer annealing, primer extending and denaturing, using the primer pairof clai 1 each having at its 3′end portion a hybridizing sequencesubstantially complementary to a region of said target nucleic acidsequence to hybridize therewith, under conditions in which each primeranneals to its target nucleotide sequence, whereby the amplificationproduct of said target nucleotide sequence is generated; and (d)performing a second-stage amplification of said amplification productgenerated from step (c) at a second annealing temperature, which is highstringent conditions, comprising at least one cycle of primer annealing,primer extending and denaturing, using the same primers as used in step(c) or a primer pair each comprising a pre-selected arbitrary nucleotidesequence corresponding to each 5′-end portion of said primers used instep (c), under conditions in which each primer anneals to the 3′- and5′-ends of said amplification product, respectively, whereby saidamplification product is re-amplified.
 45. A method for detecting DNAcomplementary to differentially expressed mRNA in two or more nucleicacid samples, wherein said method comprises reverse transcribing saidmRNA and performing an amplification reaction using primers,characterized in that at least one primer has the same structure as theprimer of claim
 1. 46. The method according to claim 45, wherein saidmethod is performed using two stage amplifications, which comprises: (a)providing a first sample of nucleic acids representing a firstpopulation of mRNA transcripts and a second sample of nucleic acidsrepresenting a second population of mRNA transcripts; (b) separatelycontacting each of said first nucleic acid sample and said secondnucleic acid sample with a first primer of claim 1, in which the 3′-endportion of said first primer comprises a hybridizing nucleotide sequencesubstantially complementary to a first site in said differentiallyexpressed mRNA to hybridize therewith, under conditions sufficient fortemplate driven enzymatic deoxyribonucleic acid synthesis to occur; (c)reverse transcribing said differentially expressed mRNA to which saidfirst primer hybridizes to produce a first population of first cDNAstrands that are complementary to said differentially expressed mRNA insaid first nucleic acid sample to which said first primer hybridizes,and a second population of first cDNA strands that are complementary tosaid differentially expressed mRNA in said second nucleic acid sample towhich said first primer hybridizes; (d) purifying and quantifying eachof said first and second populations of first cDNA strands; (e)performing a first-stage amplification of each of said first and secondpopulation of first DNA strands obtained from step (d) at a firstannealing temperature comprising at least one cycle of primer annealing,primer extending and denaturing, using a second primer of claim 1 havingat its 3 end portion a hybridizing sequence substantially complementaryto a second site in said first and second populations of first cDNAstrands, under conditions in which said second primer anneals to saidsecond site in each population of said first cDNA strands, whereby firstand second populations of second cDNA strands are generated; (f)performing a second-stage amplification of each second cDNA strandgenerated from step (e) at a second annealing temperature, which is highstringent conditions, comprising at least two cycles of primerannealing, primer extending and denaturing, using the same first andsecond primers as used in steps (b) and (e), respectively, or a primerpair each comprising a pre-selected arbitrary nucleotide sequencecorresponding to each 5′-end portion of said first and second primersused in steps (b) and (e), respectively, under conditions in which eachprimer anneals to the 3′- and 5′-end sequences of each second cDNAstrand, respectively, whereby amplification products of said second cDNAstrands are generated, and (g) comparing the presence or level ofindividual amplification products in said first and second populationsof amplification products obtained from step (f).
 47. A method forrapidly amplifying a target cDNA fragment comprising a cDNA regioncorresponding to the 3′-end region of an mRNA, wherein said methodcomprises reverse transcribing said mRNA and performing an amplificationreaction using primers, characterized in that at least one primer hasthe same structure as the primer of claim
 1. 48. The method according toclaim 47, wherein said method is performed using two stageamplifications, which comprises: (a) contacting mRNAs with a firstprimer of claim 1, in which the 3′-end portion of said primer comprisesa hybridizing nucleotide sequence substantially complementary to poly Atails of said mRNAs to hybridize therewith, under conditions sufficientfor template driven enzymatic deoxyribonucleic acid synthesis to occur;(b) reverse transcribing said mRNAs to which said first primerhybridizes to produce a population of first cDNA strands that arecomplementary to said mRNAs to which said first primer hybridizes; (c)performing a first-stage amplification of said first cDNA strands at afirst annealing temperature comprising at least one cycle of primerannealing, primer extending and denaturing, using a second primer ofclaim 1 having at its 3′-end portion a gene-specific hybridizingnucleotide sequence substantially complementary to a site in one of saidfirst cDNA strands to hybridize therewith, under conditions in whichsaid second primer anneals to a gene-specific site on one of said firstcDNA strands, whereby a gene-specific second cDNA strand is generated;and (d) performing a second-stage amplification of said gene-specificsecond cDNA strand generated from step (c) at a second annealingtemperature, which is high stringent conditions, comprising at least twocycles of primer annealing, primer extending and denaturing, using thesame first and second primers as used in steps (a) and (c),respectively, or a primer pair each comprising a pre-selected arbitrarynucleotide sequence corresponding to each 5′-end portion of said firstand second primers used in steps (a) and (c), respectively, underconditions in which each primer anneals to the 3′- and 5′-end sequencesof a gene-specific second cDNA strand, respectively, whereby anamplification product of a gene-specific cDNA strand is generated.
 49. Amethod for rapidly amplifying a target DNA fragment comprising a cDNAregion corresponding to the 5′-end region of an mRNA, wherein saidmethod comprises reverse transcribing said mRNA and performing anamplification reaction using primers, characterized in that at least oneprimer has the same structure as the primer of claim
 1. 50. The methodaccording to claim 49, wherein said method is performed using two stageamplifications, which comprises: (a) contacting mRNAs with anoligonucleotide dT primer or random primer as a cDNA synthesis primerunder conditions sufficient for template driven enzymaticdeoxyribonucleic acid synthesis to occur, in which said cDNA synthesisprimer comprises a hybridizing nucleotide sequence substantiallycomplementary to a region of an mRNA to hybridize therewith; (b) reversetranscribing said mRNAs, using a reverse transcriptase, to which saidcDNA synthesis primer hybridizes to produce a population of first cDNAstrands that are complementary to said mRNAs to which said cDNAsynthesis primer hybridizes, whereby mRNA-cDNA intermediates aregenerated; (c) permitting cytosine residues to be tailed at the 3′-endsof said first cDNA strands in the form of said mRNA-cDNA intermediatesby the terminal transferase reaction of reverse transcriptase; (d)contacting the cytosine tails at the 3′-ends of said first cDNA strandsgenerated from step (c) with an oligonucleotide which comprises a 3′-endportion and a 5′-end portion separated by a group of universal base ornon-discriminatory base analog, wherein the 3′-end portion comprises atleast three guanine residues at its 3′-end to hybridize with saidcytosine tails at the 3′-ends of said first cDNA strands and the 5′-endportion comprises a pre-selected arbitrary nucleotide sequence, underconditions in which said 3-end portion of said oligonucleotide ishybridized to said cytosine tails; (e) extending the tailed 3′-ends ofsaid first cDNA strands to generate an additional sequence complementaryto said oligonucleotide using reverse transcriptase, in which saidoligonucleotide serves as a template in the extension reaction, wherebyfull-length first cDNA strands are extended; (f) performing afirst-stage amplification of said full-length first cDNA strandsobtained from step (e) at a first annealing temperature, which comprisesthe steps of: (i) at least one cycle of primer annealing, primerextending and denaturing using a first primer comprising a nucleotidesequence substantially complementary to the 3′-end sequences of saidfull-length first cDNA strands under conditions in which said firstprimer anneals to said full-length first cDNA strands, under conditionsin which said first primer anneals to the 3′- ends of said full-lengthfirst cDNA strands, whereby full-length second cDNA strands aregenerated; (ii) at least one cycle of primer annealing, primer extendingand denaturing using a second primer of claim 1 having at its 3′-endportion a gene-specific hybridizing sequence substantially complementaryto a region on one of said full-length second cDNA strands to hybridizetherewith, under conditions in which said second primer anneals to agene-specific site on one of said full-length second cDNA strands,whereby a gene-specific cDNA strand is generated; and (g) performing asecond-stage amplification of said gene-specific cDNA strand at a secondannealing temperature, which is high stringent conditions, comprising atleast two cycles of primer annealing, primer extending and denaturing,using the same first and second primers as used in steps (f)-(i) and(f)-(ii), respectively, or a primer pair each comprising a nucleotidesequence corresponding to each 5′-end portion of said first and secondprimers as used in steps (f)-(i) and (f)-(ii), respectively, underconditions in which each primer anneals to the 3′- and 5′-end sequencesof a gene-specific cDNA strand, respectively, whereby an amplificationproduct of a gene-specific cDNA strand is generated.
 51. A method foramplifying a population of full-length double-stranded cDNAscomplementary to mRNAs, wherein said method comprises reversetranscribing said mRNA and performing an amplification reaction usingprimers, characterized in that at least one primer has the samestructure as the primer of claim
 1. 52. The method according to claim51, wherein said method comprises: (a) contacting said mRNAs with afirst primer of claim 1, in which the 3′-end portion of said firstprimer has a hybridizing nucleotide sequence substantially complementaryto poly A tails of said mRNAs to hybridize therewith, under conditionssufficient for template driven enzymatic deoxyribonucleic acid synthesisto occur; (b) reverse transcribing said mRNAs, using a reversetranscriptase, to which said first primer hybridizes to produce saidpopulation of first cDNA strands that are complementary to said mRNAs towhich said primer hybridizes, whereby mRNA-cDNA intermediates aregenerated; (c) permitting cytosine residues to be tailed at the 3′-endsof said first cDNA strands in the form of said mRNA-cDNA intermediatesby the terminal transferase reaction of reverse transcriptase; (d)contacting the cytosine tails at the 3′-ends of said first cDNA strandsgenerated from step (c) with an oligonucleotide which comprises a 3′-endportion and a 5′-end portion separated by a group of universal base ornon-discriminatory base analog, wherein the 3′-end portion comprises atleast three guanine residues at its 3′-end to hybridize with saidcytosine tails at the 3′-ends of said first cDNA strands and the 5′-endportion comprises a pre-selected arbitrary nucleotide sequence, underconditions in which said 3-end portion of said oligonucleotide ishybridized to said cytosine tails; (e) extending the tailed 3′-ends ofsaid first cDNA strands to generate an additional sequence complementaryto said oligonucleotide using reverse transcriptase, in which saidoligonucleotide serves as a template in the extension reaction, wherebyfull-length first cDNA strands are extended; and (f) performing anamplification of said full-length first cDNA strands generated from step(e) comprising at least two cycles of primer annealing, primer extendingand denaturing, 4 0 using a primer pair each comprising a nucleotidesequence corresponding to the same first primer and oligonucleotide asused in steps (a) and (d), respectively, or a primer pair eachcomprising a nucleotide sequence corresponding to each 5′-end portion ofsaid first primer and oligonucleotide used in steps (a) and (d),respectively, under conditions in which each primer anneals to the 3′-and 5′-end sequences of said full-length first cDNA strands,respectively, whereby amplification products of full-length cDNA strandscomplementary to said mRNAs are generated.
 53. A method for amplifying5′-enriched double-stranded cDNAs complementary to mRNAs, wherein saidmethod comprises comprising reverse transcribing said mRNAs andperforming an amplification reaction using primers, characterized inthat at least one primer has the same structure as the primer ofclaim
 1. 54. The method according to claim 53, wherein said methodcomprises: (a) contacting said mRNAs with a first primer of claim 1under conditions sufficient for template driven enzymaticdeoxyribonucleic acid synthesis to occur, wherein the 3′-end portion ofsaid first primer has at least six random nucleotide sequences; (b)performing the steps (b)-(e) of claim 52, whereby 5′-enriched first cDNAstrands are extended; (c) performing an amplification of said5′-enriched first cDNA strands generated from step (b) comprising atleast two cycles of primer annealing, primer extending and denaturing,using a primer pair each comprising a nucleotide sequence correspondingto each 5′-end portion of said primer and oligonucleotide used in steps(a) and (b), respectively, under conditions in which each primer annealsto the 3′- and 5′-end sequences of said 5′-enriched first cDNA strands,respectively, whereby amplification products of 5′-enriched cDNA strandsare generated.
 55. A method for amplifying more than one targetnucleotide sequence simultaneously using more than one pair of primersin the same reaction, wherein said method comprises performing anamplification reaction using primers, characterized in that at least oneprimer has the same structure as the primer of claim
 1. 56. The methodaccording to claim 55, wherein said method is performed using two stageamplifications, which comprises: (a) performing a first-stageamplification of more than one target nucleotide sequence at a firstannealing temperature comprising at least two cycles of primerannealing, primer extending and denaturing, using the primer pairs ofclaim 1 in which its 3′end portion each of each primer pair has ahybridizing nucleotide sequence substantially complementary to a regionof said target nucleic acid sequence to hybridize therewith, underconditions in which each of said primer pairs anneals to its targetnucleotide sequence, whereby the amplification products of targetnucleotide sequences are generated; and (b) performing a second-stageamplification of said amplification products generated from step (a) ata second annealing temperature, which is high stringent conditions,comprising at least one cycle of primer annealing, primer extending anddenaturing, using the same primer pairs as used in step (a) or primerpairs each comprising a pre-selected arbitrary nucleotide sequencecorresponding to each 5′-end portion of said primer pairs used in step(a), under conditions in which each of each primer pair anneals to the3′- and 5′-end sequences of said amplification products generated fromstep (a), respectively, whereby said amplification products arere-amplified in the same reaction.
 57. A method for producing a DNAfingerprint of gDNA, wherein said method comprises performing anamplification reaction using primers, characterized in that at least oneprimer has the same structure as the primer of claim
 1. 58. The methodaccording to claim 57, wherein said method is performed using two stageamplifications, which comprises: (a) performing a first-stageamplification of said DNA fingerprint, which is a set of discrete DNAsegments characteristic of genome, from said gDNA at a first annealingtemperature comprising at least two cycles of primer annealing, primerextending and denaturing, using the primer or the primer pair of claim1, wherein each primer has at its 3′-end portion an arbitrary nucleotidesequence substantially complementary to sites on said gDNA to hybridizetherewith, under conditions in which said primer or said primer pairanneals to said gDNA, whereby said set of discrete DNA segmentscharacterized as a DNA fingerprint is produced; and (b) performing asecond-stage amplification of said set of discrete DNA segmentsgenerated from step (a) at a second annealing temperature, which is highstringent conditions, comprising at least one cycle of primer annealing,primer extending and denaturing, using the same primer or primer pair asused in step (a) or a primer or a primer pair each comprising anucleotide sequence corresponding to each 5′-end portion of said primeror primer pair used in step (a), under conditions in which said primeror each of said primer pair anneals to the 3′- and 5′-end sequences ofsaid set of discrete DNA segments generated from step (a), respectively,whereby said set of discrete DNA segments is re-amplified.
 59. A methodfor producing a RNA fingerprint of an mRNA sample, wherein said methodcomprises reverse transcribing and performing an amplification reactionusing primers, characterized in that at least one primer has the samestructure as the primer of claim
 1. 60. The method according to claim59, wherein said method is performed using two stage amplifications,which comprises: (a) contacting said mRNA sample with a first primer ofclaim 1, in which said first primer has a hybridizing nucleotidesequence substantially complementary to poly A tails of said mRNA sampleto hybridize therewith, under conditions sufficient for template drivenenzymatic deoxyribonucleic acid synthesis to occur; (b) reversetranscribing said mRNA sample to which said first primer hybridizes toproduce a population of first cDNA strands that are complementary tosaid mRNA sample to which said first primer hybridizes; (c) performing afirst-stage amplification of said population of first cDNA strandsgenerated from step (b) at a first annealing temperature comprising atleast one cycle of primer annealing, primer extending and denaturing,using a second primer or primer pair of claim 1, wherein each primer hasat its 3′-end portion an arbitrary nucleotide sequence substantiallycomplementary to sites on said first cDNA strands to hybridizetherewith, under conditions in which said primer or primer pair annealsto said mRNA sample, whereby a set of discrete cDNA segmentscharacterized as a RNA fingerprint is produced; and (d) performing asecond stage amplification of said set of discrete cDNA segmentsgenerated from step (c) at a second annealing temperature which is highstringent conditions, comprising at least one cycle of primer annealing,primer extending and denaturing, using the same primer or primer pair asused in step (c) or a primer or primer pair each comprising a nucleotidesequence corresponding to each 5′-end portion of said primer or primerpair used in step (c), under conditions in which said primer or each ofsaid primer pair anneals to the 3′- and 5′-end sequences of said set ofdiscrete cDNA segments generated from step (c), respectively, wherebysaid set of discrete cDNA segments is re-amplified.
 61. A method foridentifying conserved homology segments in a multigene family from anmRNA sample, wherein said method comprises reverse transcribing andperforming an amplification reaction using primers, characterized inthat at least one primer has the same structure as the primer ofclaim
 1. 62. The method according to claim 61, wherein said method isperformed using two stage amplifications, which comprises: (a)contacting said mRNA sample with a first primer of claim 1, in whichsaid first primer has a hybridizing nucleotide sequence substantiallycomplementary to poly A tails of said mRNA sample to hybridizetherewith, under conditions sufficient for template driven enzymaticdeoxyribonucleic acid synthesis to occur; (b) reverse transcribing saidmRNA sample to which said first primer hybridizes to produce apopulation of first cDNA strands that are complementary to said mRNAsample to which said first primer hybridizes; (c) performing afirst-stage amplification of said population of first cDNA strandsgenerated from step (b) at a first annealing temperature comprising atleast one cycle of claim 1 having at its 3′end portion a hybridizingsequence substantially complementary to a consensus sequence or adegenerate sequence encoding amino acid sequence of a conserved homologysegment on said first cDNA strands to hybridize therewith, underconditions in which said second primer anneals to said consensussequence or degenerate sequence of first cDNA strands, whereby 3′-endcDNA segments having said consensus sequence or degenerate sequence aregenerated; and (d) performing a second stage amplification of said3′-end cDNA segments generated from step (c) at a second annealingtemperature which is high stringent conditions, comprising at least twocycles of primer annealing, primer extending and denaturing, using thesame first and second primers as used in steps (a) and (c) or a primerpair each comprising a nucleotide sequence corresponding to each 5′-endportion of said first and second primers used in steps (a) and (c),respectively, under conditions in which each primer anneals to the 3′-and 5′-end sequences of said 3′-end cDNA segments, respectively, wherebysaid 3′-end conserved homology cDNA segments are amplified.
 63. Themethod according to claim 61, wherein said method is performed using twostage 10 amplifications, which comprises: (a) performing steps of(a)-(e) of claim 52, whereby full-length cDNA strands are generated; (b)performing a first-stage amplification of said full-length first cDNAstrands obtained from step (a) at a first annealing temperature, whichcomprises the steps of: (i) at least one cycle of primer annealing,primer extending and denaturing using a first primer comprising anucleotide sequence substantially complementary to the 3′-end sequencesof said full-length first cDNA strands under conditions in which saidfirst primer anneals to said full-length first cDNA strands, underconditions in which said first primer anneals to the 3′- ends of saidfull-length first cDNA strands, whereby full-length second cDNA strandsare generated; and (ii) at least one cycle of primer annealing, primerextending and denaturing using a second primer of claim 1 having at its3′end portion a hybridizing sequence substantially complementary to aconsensus sequence or a degenerate sequence encoding amino acid sequenceof a conserved homology segment on said full-length second cDNA strandsto hybridize therewith, under conditions in which said second primeranneals to said consensus sequence or degenerate sequence of full-lengthsecond cDNA strands, whereby 5′-end cDNA segments having said consensussequence or degenerate sequence are generated; and (c) performing asecond stage amplification of said 5′-end cDNA segments generated fromstep (b) at a second annealing temperature which is high stringentconditions, comprising at least two cycles of primer annealing, primerextending and denaturing, using the same first and second primers asused in steps (b)-(i) and (b)-(ii), respectively, or a primer pair eachcomprising a nucleotide sequence corresponding to each 5′-end portion ofsaid first and second primers used in steps (b)-(i) and (b)-(ii),respectively, under conditions in which each primer anneals to the 3′-and 5′-end sequences of said 5′-end cDNA segments, respectively, wherebysaid 5′-end conserved homology cDNA segments are amplified.
 64. A methodfor identifying conserved homology segments in a multigene family fromgDNA, wherein said method comprises performing an amplification reactionusing primers, characterized in that at least one primer has the samestructure as the primer of claim
 1. 65. The method according to claim64, wherein said method is performed using two stage amplifications,which comprises: (a) performing a first-stage amplification of saidconserved homology segments from said gDNA at a first annealingtemperature comprising at least two cycles of primer annealing, primerextending and denaturing, using the primer or the primer pair of claim1, wherein each primer has at its 3′ end portion a hybridizing sequencesubstantially complementary to a consensus sequence or a degeneratesequence encoding amino acid sequence of a conserved homology segment onsaid gDNA to hybridize therewith, under conditions in which said primeror said primer pair anneals to said consensus sequence or degeneratesequence of gDNA, whereby genomic DNA segments having said consensussequence or degenerate sequence are generated; and (b) performing asecond-stage amplification of said genomic DNA segments generated fromstep (a) at a second annealing temperature, which is high stringentconditions, comprising at least one cycle of primer annealing, primerextending and denaturing, using the same primer or primer pair as usedin step (a) or a primer or a primer pair each comprising a nucleotidesequence corresponding to each 5′-end portion of said primer or primerpair used in step (a), under conditions in which said primer or each ofsaid primer pair anneals to the 3′- and 5′-end sequences of said genomicDNA segments generated from step (a), respectively, whereby saidconserved homology genomic segments are amplified.
 66. A method foridentifying a nucleotide variation in a target nucleic acid, whereinsaid method comprises performing an amplification reaction usingprimers, characterized in that at least one primer has the samestructure as the primer of claim
 1. 67. The method according to claim66, wherein said method is performed using two stage amplifications,which comprises: (a) performing a first-stage amplification to produce afirst DNA strand complementary to said target nucleic acid includingsaid nucleotide variation at a first annealing temperature comprising atleast one cycle of primer annealing, primer extending and denaturing,using a first primer of claim 1 having at its 3′-end portion ahybridizing sequence substantially complementary to a pre-selectedsequence at a first site of said target nucleic acid to hybridizetherewith, wherein each of said first primer and said first sitecomprises an interrogation position corresponding to said nucleotidevariation, whereby said first DNA strand complementary to said targetnucleic acid including said nucleotide variation is generated when saidinterrogation position is occupied by the complementary nucleotide ofsaid first primer to its corresponding nucleotide of said first site;and (b) performing a second-stage amplification of said first DNA strandgenerated from step (a) at a second annealing temperature, which is highstringent conditions, comprising the steps: (i) at least one cycle ofprimer annealing, primer extending and denaturing using a second primerof claim 1 having at its 3′-end portion a hybridizing sequencesubstantially complementary to a pre-selected sequence at a second siteof said target nucleic acid to hybridize therewith under conditions inwhich said second primer anneals to said second site of the targetnucleic acid, whereby a second DNA strand complementary to said firstDNA strand including said nucleotide variation is generated; and (ii) atleast one cycle of primer annealing, primer extending and denaturingusing the same first and second primers as used in steps (a) and (b)-(i)or a primer pair each having a hybridizing sequence complementary orcorresponding to the 3′- and 5′-ends of said second DNA strand generatedfrom step (b)-(i) to hybridize therewith, under conditions in which eachprimer anneals to the 3′- and 5′-end sequences of said second DNAstrand, respectively, whereby said second DNA strand which comprisessaid first and second sites of said target nucleic acid at its 3′-and5′-ends is amplified so that a short target nucleotide segmentcorresponding to said second DNA strand containing said nucleotidevariation is generated.
 68. The method according to claim 66, whereinsaid method is performed using two individual amplifications of a firstand a second amplifications in which said second amplification isperformed using two stage amplifications, which comprises: (a)performing said first amplification to produce a short DNA strandfragment containing said nucleotide variation between its endscomprising at least two cycles of primer annealing, primer extending anddenaturing, using a primer pair each primer comprising a hybridizingsequence substantially complementary to a pre-selected sequence at asite of said target nucleic acid under conditions that said nucleotidevariation is positioned between said pre-selected sequences, in which atleast one primer of said primer set is the primer of claim 1 having atits 3′-end portion said hybridizing sequence, whereby said short DNAstrand fragment containing said nucleotide variation between its ends isamplified; (b) performing a first-stage amplification of said secondamplification to produce a first DNA strand complementary to said shortDNA strand fragment including said nucleotide variation at a firstannealing temperature comprising at least one cycle of primer annealing,primer extending and denaturing, using a first primer of claim lhavingat its 3′-end portion a hybridizing sequence substantially complementaryto a pre-selected sequence at a first site of said target nucleic acidto hybridize therewith, wherein each of said first primer and said firstsite comprises an interrogation position corresponding to saidnucleotide variation, whereby said first DNA strand complementary tosaid target nucleic acid including said nucleotide variation isgenerated when said interrogation position is occupied by thecomplementary nucleotide of said first primer to its correspondingnucleotide of said first site; and (c) performing a second-stageamplification of said second amplification of said first DNA strandgenerated from step (a) at a second annealing temperature, which is highstringent conditions, comprising at least one cycle of primer annealing,primer extending and denaturing using a primer pair in which amongstsaid primer pair one is the same as said primer of claim 1 used in step(a) the other is the same as said first primer used in step (b), or aprimer pair each having a hybridizing sequence complementary orcorresponding to the 3′- and 5′-ends of said first DNA strand generatedfrom step (b) to hybridize therewith, under conditions in which eachprimer anneals to the 3′- and 5′-end sequences of said first DNA strand,respectively, whereby said first DNA strand is amplified so that a shorttarget nucleotide segment corresponding to said first DNA strandcontaining said nucleotide variation is generated.
 69. A method formutagenesis in a target nucleic acid, comprising performing anamplification reaction using primers, characterized in that at least oneprimer has the same structure as the primer of claim
 1. 70. The methodaccording to claim 69, wherein said mutagenesis is site-directed anduses two stages amplifications, comprising the steps of: (a) performinga first-stage amplification of said target nucleic acid sequence at afirst annealing temperature comprising at least two cycles of primerannealing, primer extending and denaturing, using a primer pair of claim1 each having at its 3′end portion a hybridizing sequence substantiallycomplementary to a region of said target nucleic acid sequence tohybridize therewith, wherein said hybridizing sequence has at least onemismatch nucleotide to generate site-directed mutation, under conditionsin which said primer or primer pair anneals to its target nucleotidesequence, whereby an amplification product containing site-directedmutation site is generated; and (b) performing a second-stageamplification of said amplification product generated from step (a) at asecond annealing temperature, which is high stringent conditions,comprising at least one cycle of primer annealing, primer extending anddenaturing, using the same primers as used in step (a) or a primer paireach comprising a pre-selected arbitrary nucleotide sequencecorresponding to each 5′-end portion of said primers used in step (a),under conditions in which each primer anneals to the 3′- and 5′-ends ofsaid amplification product, respectively, whereby said amplificationproduct containing site-directed mutation site is re-amplified.
 71. Akit for nucleic acid amplification, which comprises the annealingcontrol primer or annealing control primer set described in claim 40.72. A kit for selective amplification of a target nucleic acid sequencefrom DNA, which comprises the annealing control primer or annealingcontrol primer set described in claim
 42. 73. A kit for selectiveamplification of a target nucleic acid sequence from mRNA, whichcomprises the annealing control primer or annealing control primer setdescribed in claim
 44. 74. A kit for detecting DNA complementary todifferentially expressed mRNA, which comprises the annealing controlprimer or annealing control primer set described in claim
 46. 75. A kitfor rapidly amplifying a target cDNA fragment comprising a cDNA regioncorresponding to the 3′-end region of an mRNA, which comprises theannealing control primer or annealing control primer set described inclaim
 48. 76. A kit for rapidly amplifying a target cDNA fragmentcomprising a cDNA region corresponding to the 5′-end region of an mRNA,which comprises the annealing control primer or annealing control primerset described in claim
 50. 77. A kit for amplifying a population offull-length double-stranded cDNAs complementary to mRNAs, whichcomprises the annealing control primer or annealing control primer setdescribed in claim
 52. 78. A kit for amplifying 5′-enricheddouble-stranded cDNAs complementary to the 5′-end regions of mRNAs,which comprises the annealing control primer or annealing control primerset described in claim
 54. 79. A kit for amplifying more than one targetnucleotide sequence simultaneously, which comprises the annealingcontrol primer or annealing control primer set described in claim 56.80. A kit for producing a DNA fingerprint by use of gDNA, whichcomprises the annealing control primer or annealing control primer setdescribed in claim
 58. 81. A kit for producing a RNA fingerprint by useof mRNA, which comprises the annealing control primer or annealingcontrol primer set described in claim
 60. 82. A kit for identifying aconserved homology segment in a multigene family by use of mRNA, whichcomprises the annealing control primer or annealing control primer setdescribed in claim 62 or
 63. 83. A kit for identifying a conservedhomology segment in a multigene family by use of gDNA, which comprisesthe annealing control primer or annealing control primer set describedin claim
 65. 84. A kit for identifying a nucleotide variation in atarget nucleic acid, which comprises the annealing control primer orannealing control primer set described in claim 67 or
 68. 85. A kit formutagenesis in a target nucleic acid, which comprises the annealingcontrol primer or annealing control primer set described in claim 70.